Yersinia pestis vaccine

ABSTRACT

The present invention encompasses a recombinant  Yersinia pestis  bacterium and a vaccine comprising a recombinant  Yersinia pestis  bacterium.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. provisional applicationSer. No. 61/294,727, filed Jan. 13, 2010, which is hereby incorporatedby reference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under Al057885 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The invention encompasses a recombinant Yersinia bacterium.

BACKGROUND OF THE INVENTION

Plague remains one of the most feared infectious diseases in humans. Theetiological agent of the disease, Yersinia pestis, is disseminated byfleas and infects both humans and rodents. Y. pestis rapidly invadesfrom the infection site into the lymphatic system and circulation, toproduce the systemic and often fatal disease. Plague is endemic in manyareas of the world, including the western United States. Globally about2000 cases of plague are reported to the World Health Organization eachyear. Most of these cases are the bubonic form of the disease, usually aconsequence of the transmission of bacteria to humans via bites fromfleas that have previously fed on infected rodents. Although the mostcommon mode of transmission is the flea bite, oral transmission canoccur, often the result of an animal (polecat, weasel, ferret, cat)feeding on an infected mouse or other small rodent. Although lesscommon, contact with domestic cats that have been exposed to Y. pestisis an important transmission mode because of the higher than averageincidence of pneumonic plague that occurs in these cases. More rarely,cases of pneumonic plague are reported that are characterized by a shortincubation period of 2 to 3 days and a high rate of mortality, even iftreated. Pneumonic plague can be transmitted person-to-person oranimal-to-person via the inhalation of contaminated air droplets.Pneumonic plague is the most likely form to be encountered if Y. pestisis used as a biological weapon.

Recent efforts to create a safe and effective pneumonic plague vaccinehave focused on the development of recombinant subunit vaccines thatelicit antibodies against two well characterized Y. pestis antigens, theF1 capsule and the virulence protein LcrV. In the past, live attenuatedvaccine strains were generated by selection, rather than precise geneticmanipulation, thus raising concern about their genetic composition andstability. An early live plague vaccine strain, EV76, has been used insome countries. However, EV76 has been known to cause disease inprimates, raising questions about its suitability as a human vaccine.There is a need in the art, therefore, for a live plague vaccine usingan adequately attenuated, rationally designed Y. pestis strain. Thiswill provide the advantage of simultaneous priming against more than oneantigen, thereby greatly enhancing the likelihood of broad-basedprotection.

SUMMARY OF THE INVENTION

One aspect of the present invention encompasses a recombinant Yersiniabacterium, wherein the bacterium comprises a regulated attenuationmutation.

Another aspect of the present invention encompasses a vaccine. Thevaccine comprises a recombinant Yersinia bacterium, wherein thebacterium comprises a regulated attenuation mutation.

Yet another aspect of the present invention encompasses a method foreliciting both a humoral and a cellular immune response to Yersinia in ahost. The method comprises administering a vaccine to a subject. Thevaccine generally comprises a recombinant Yersinia bacterium, whereinthe bacterium comprises a regulated attenuation mutation.

Still another aspect of the present invention encompasses a method foreliciting a protective immune response against bubonic and pneumonicplaque. The method comprises administering a vaccine to a subject. Thevaccine generally comprises a recombinant Yersinia pestis bacterium,wherein the bacterium comprises a regulated attenuation mutation.

Other aspects and iterations of the invention are described morethoroughly below.

BRIEF DESCRIPTION OF THE FIGS.

The application file contains at least one photograph executed in color.Copies of this patent application publication with color photographswill be provided by the Office upon request and payment of the necessaryfee.

FIG. 1 depicts a schematic chromosome structure of Y. pestis KIM6+,χ10003 (ΔrelA233), χ10004 (ΔrelA233ΔspoT85) and χ10019(ΔrelA233ΔspoT85ΔlacZ::TT araC P_(BAD) spoT).

FIG. 2 depicts TLC analysis of (p)ppGpp synthesis in Y. pestis KIM6+ andΔrelA ΔspoT mutants during amino acid and carbon starvation. Totalintracellular nucleotides were extracted from Y. pestis culturesuniformly labeled with [³²P] H₃PO₄. Cells were grown in modified PMH2medium lacking L-phenylalanine for amino acid starvation (A) and inmodified PMH2 medium without glucose for carbon starvation (B).

FIG. 3 depicts a schematic chromosome structure of Y. pestis KIM6+,χ10021 (spoT412:: 3× flag-kan), χ10019 (ΔrelA233 ΔspoT85 ΔlacZ516::TTaraC P_(BAD) spoT) and χ10022 (ΔrelA233 ΔspoT85 ΔlacZ516 ΩTT araCP_(BAD) spoT413:: 3×flag-kan).

FIG. 4 depicts measurement of SpoT expression M, protein marker; 1, Y.pestis KIM6+; 2, χ10021; 3, χ10022 (without arabinose); 4, χ10022 (with0.05% arabinose); 5, χ10022 (with 0.1% arabinose); 6, χ10022 (with 0.15%arabinose); 7, χ10022 (with 0.2% arabinose); 8, χ10022 (with 0.3%arabinose).

FIG. 5 depicts growth of Y. pestis strains in HIB medium at differenttemperatures (A) Growth curve at 26° C.; (B) Growth curve at 37° C. ●,Y. pestis KIM5+; ▪, χ10003(pCD1Ap) (ΔrelA233) ▴, χ10004(pCD1Ap)(ΔrelA233ΔspoT85); ▾, χ10019(pCD1Ap) (ΔrelA233 ΔspoT85 ΔlacZ::TT araCP_(BAD) spoT) without arabinose; ♦, χ10019(pCD1Ap) (ΔrelA233 ΔspoT85ΔlacZ::TT araC P_(BAD) spoT) with 0.05% arabinose.

FIG. 6 depicts the analysis of virulence factor expression and secretionin Y. pestis KIM5+ and mutants. (A) Evaluation of virulence factortranscription by semi-quantitative RT-PCR. (B) Measurement of secretedvirulence factors in culture supernatants by western blotting. Secretedproteins were collected from the culture medium following the removal ofbacterial cells. Proteins were separated by SDS-PAGE and detected bywestern blotting. For each sample, the same amount of total protein wasloaded.

FIG. 7 depicts 2-DE gels (A) Comparing differential protein expressionbetween KIM5+ (wild-type Y. pestis) and χ10004-pCD1Ap (ΔrelA233 ΔspoT85)at 26° C. (B) Comparing differential protein expression between KIM5+(wild-type Y. pestis) and χ10004-pCD1Ap (ΔrelA233 ΔspoT85) at 37° C.

FIG. 8 depicts the survival of Swiss Webster mice (3 mice per strain)infected s.c. with Y. pestis KIM5+ (black circles), χ10003(pCD1Ap)(black squares), χ10004(pCD1Ap) (black triangles) and χ10019(pCD1Ap)cultured with 0.05% arabinose in vitro (black diamonds).The experimentwas performed twice with similar results.

FIG. 9 depicts the kinetics of infection with Y. pestis KIM5+ (black) orχ10004(pCD1Ap) (white) in mouse tissues. Groups of nine mice wereinoculated s.c., and at various times CFU per organ in the blood (A),lungs (B), spleens (C) and livers (D) were determined for 3 mice pergroup. Error bars represent standard deviation.

FIG. 10 depicts the antibody response in sera of mice inoculated with Y.pestis KIM5+ or χ10004(pCD1Ap). A Y. pestis whole cell lysate was usedas the coating antigen. (A) Serum IgG responses. (B) Serum IgG1 andIgG2a responses. *, the P value was less than 0.01; **, the P value wasless than 0.05.

FIG. 11 depicts mouse survival after Y. pestis KIM5+ challenge. (A)Swiss Webster mice vaccinated s.c. with 2.5×10⁴ CFU of χ10004(pCD1Ap)and a were challenged with 1.5×10⁵ CFU of Y. pestis KIM5+ via the s.c.route. (B) Swiss Webster mice vaccinated s.c. with 2.5×10⁴ CFU ofχ10004(pCD1Ap) were challenged via the i.n. route with 2×10⁴ CFU of Y.pestis KIM5+. Immunization provided significant protection against bothchallenge routes (P<0.001). For each experiment, there were 10 mice inthe vaccinated group and 4 mice in the control group.

FIG. 12 depicts IL-10 production in sera of mice inoculated with Y.pestis KIM5+ or χ10004(pCD1Ap). *, the P value was less than 0.01; **,the P value was less than 0.05.

FIG. 13 depicts the structure of chromosomal region of Y. pestis strainsKIM6+, χ10010, and χ10017.

FIG. 14 depicts Crp synthesis and growth of Y. pestis mutants. (A)Measurement of Crp synthesis in Y. pestis KIM5+, χ10010 (crp18) andχ10017 (araC P_(BAD) crp). Strains were grown in HIB at 37° C. overnightand Crp synthesis was detected by western blot using anti-Crp sera. M,protein marker. (B) Growth of Y. pestis strains in HIB medium at 26° C.or 37° C. ●, Y. pestis KIM5+; ▪, χ10010(pCD1Ap) (Δcrp); ▴χ10017(pCD1Ap)(araC P_(BAD) crp) without arabinose; ▾, χ10017(pCD1Ap) with 0.05%arabinose.

FIG. 15 depicts the measurement of LcrV synthesis and secretion in Y.pestis by western blot analysis. Whole cell lysates and supernatantfractions were separated by SDS-PAGE and detected by western blotting.For each sample, equivalent amounts of protein were loaded. The araCP_(BAD) crp strain χ10017(pCD1Ap) was grown with and without 0.05%arabinose.

FIG. 16 depicts kinetics of infection with Y. pestis KIM5+ and mutantderivatives in mouse tissues. Bacteria were inoculated s.c., with1.5×10³ CFU of Y. pestis KIM5+, 4.2×10⁷ CFU of χ10010(pCD1Ap) or 3.8×10⁶CFU of χ10017(pCD1Ap) and at various times CFU per organ in the blood(A), lungs (B), spleens (C) and livers (D) were determined. Error barsrepresent standard deviations. We examined 3 mice/group/time point andthe experiment was performed twice with similar results.

FIG. 17 depicts the survival of immunized and non-immunized mice afterY. pestis KIM5+ challenge. (A) Swiss Webster mice vaccinated s.c. with3.8×10⁷ CFU of χ10010(pCD1Ap) or 2.5×10⁷ CFU of Y. pestis KIM5 (Pgm⁻)were challenged with 1.3×10⁷ CFU of Y. pestis KIM5+ via the s.c. route.(B) Swiss Webster mice vaccinated s.c. with 3.0×10⁴ CFU ofχ10017(pCD1Ap) were challenged with 1.4×10⁵ CFU of Y. pestis KIM5+ viathe s.c. route. (C) Swiss Webster mice vaccinated s.c. with 3.8×10⁷ CFUof χ10010(pCD1Ap), 3.0×10⁴ CFU of χ10017(pCD1Ap) or 3.8×10⁷ CFU ofχ10010(pCD1Ap) were challenged via the i.n. route with 1.4×10⁴ CFU of Y.pestis KIM5+. For panels A and B, survival of immunized mice wassignificantly greater than PBS controls in all experiments (P<0.001).For panel C, survival of mice immunized with χ10017(pCD1Ap) or KIM5 wassignificantly greater than mice immunized with strain χ10010(pCD1Ap) orPBS controls (P<0.001). There were 10 mice per vaccination group and 4mice per control group for each experiment. The experiment was performedtwice.

FIG. 18 depicts the IgG response in sera of mice inoculated withχ10010(pCD1Ap) or χ10017(pCD1Ap). (A) Y. pestis KIM5+ whole cell lysate(YpL) was used as the coating antigen; (B) recombinant LcrV was used asthe coating antigen. *, P<0.01.

FIG. 19 depicts Serum IgG1 and IgG2a responses to YpL and recombinantLcrV. (A) IgG1 and IgG2a antibody levels to YpL in sera of miceimmunized s.c. with χ10010(pCD1Ap); (B) IgG1 and IgG2a antibody levelsto recombinant LcrV in sera of mice subcutaneously immunized withχ10010(pCD1Ap); (C) IgG1 and IgG2a antibody levels to YpL in sera ofmice subcutaneously immunized with χ10017(pCD1Ap); (D) IgG1 and IgG2aantibody levels to recombinant LcrV in sera of mice subcutaneouslyimmunized with χ10017(pCD1Ap). *, P<0.01.

DETAILED DESCRIPTION

The present invention encompasses a recombinant Yersinia bacterium. Thebacterium generally comprises a regulated attenuation mutation. Thebacterium may also be capable of the regulated expression of at leastone nucleic acid sequence encoding an antigen of interest. The inventionfurther comprises a vaccine comprising a recombinant Yersinia bacterium,and a method of eliciting an immune response to Yersinia or anotherpathogen. In exemplary embodiments, a vaccine of the invention elicits aprotective immune response to both bubonic and pneumonic plague.

Several Yersinia species are suitable for use in the present invention.In one embodiment, a recombinant Yersinia bacterium of the invention maybe a Yersinia pestis bacterium. In another embodiment, a recombinantYersinia bacterium of the invention may be a Y. enterocoliticabacterium. In yet another embodiment, a recombinant Yersinia bacteriummay be a Y. pseudotuberculosis bacterium.

I. Regulated Attenuation

The present invention encompasses a recombinant Yersinia bacteriumcapable of regulated attenuation. “Attenuation,” as used herein, refersto the state of the bacterium wherein the bacterium has been weakenedfrom its wild-type fitness by some form of recombinant or physicalmanipulation. This includes altering the genotype of the bacterium toreduce its ability to cause disease. However, the bacterium's ability tocolonize the host and induce immune responses is, preferably, notsubstantially compromised. “Regulated attenuation,” as used herein,refers to controlling when and/or where the bacterium is attenuated in ahost. Typically, a bacterium initially colonizes the host in anon-attenuated manner, and is attenuated after several replicationcycles.

A bacterium capable of regulated attenuation typically comprises achromosomally integrated regulatable promoter. The promoter replaces thenative promoter of, and is operably linked to, at least one nucleic acidsequence encoding an attenuation protein, such that the absence of thefunction of the protein renders the bacterium attenuated. In someembodiments, the promoter is modified to optimize the regulatedattenuation.

In each of the embodiments described herein, more than one method ofattenuation may be used. For instance, a recombinant bacterium of theinvention may comprise a regulatable promoter chromosomally integratedso as to replace the native promoter of, and be operably linked to, atleast one nucleic acid sequence encoding an attenuation protein, suchthat the absence of the function of the protein renders the bacteriumattenuated, and the bacterium may comprise another method of attenuationdetailed in section I(f) below.

(a) Attenuation Protein

Herein, “attenuation protein” is meant to be used in its broadest senseto encompass any protein the absence of which attenuates a bacterium.For instance, in some embodiments, an attenuation protein may be aprotein that helps protect a bacterium from stresses encountered in thegastrointestinal tract or respiratory tract. Non-limiting examples maybe the RelA, SpoT, OmpR, Crp, RpoS, Fur, Asd and MurA proteins. In otherembodiments, the protein may be a necessary component of the cell wallof the bacterium. In still other embodiments, the protein may be listedin Section I(f) below.

The native promoter for a nucleic acid encoding at least one, two,three, four, or more than four attenuation proteins may be replaced by aregulatable promoter as described herein. In one embodiment, thepromoter for a nucleic acid encoding one of the proteins selected fromthe group comprising RelA, SpoT, OmpR, and Crp may be replaced. Inanother embodiment, the promoter for a nucleic acid encoding two, three,or four of the proteins selected from the group comprising RelA, SpoT,OmpR, and Crp may be replaced.

If the promoter of a nucleic acid encoding more than one attenuationprotein is replaced, each promoter may be replaced with a regulatablepromoter, such that the expression of a nucleic acid encoding eachattenuation protein is regulated by the same compound or condition.Alternatively, each promoter may be replaced with a differentregulatable promoter, such that the expression of each attenuationprotein encoding sequence is regulated by a different compound orcondition such as by the sugars arabinose, maltose, rhamnose or xylose.

(b) Regulatable Promoter

Generally speaking, the native promoter of a nucleic acid encoding anattenuation protein may be replaced with a regulatable promoter operablylinked to the nucleic acid sequence encoding an attenuation protein. Theterm “operably linked,” as used herein, means that expression of anucleic acid is under the control of a promoter with which it isspatially connected. A promoter may be positioned 5′ (upstream) of thenucleic acid under its control. The distance between the promoter and anucleic acid to be expressed may be approximately the same as thedistance between that promoter and the native nucleic acid sequence itcontrols. As is known in the art, variation in this distance may beaccommodated without loss of promoter function but can also be used tomodulate (e.g. to increase or decrease) promoter function.

The regulatable promoter used herein generally allows transcription ofthe nucleic acid sequence encoding the attenuation protein while in apermissive environment (i.e. in vitro growth), but ceases transcriptionof the nucleic acid sequence encoding an attenuation protein while in anon-permissive environment (i.e. during growth of the bacterium in ananimal or human host). For instance, the promoter may be responsive to aphysical or chemical difference between the permissive andnon-permissive environment. Suitable examples of such regulatablepromoters are known in the art and detailed in Section II below.

In some embodiments, the promoter may be responsive to the level ofarabinose in the environment. In other embodiments, the promoter may beresponsive to the level of maltose, rhamnose, or xylose in theenvironment. The promoters detailed herein are known in the art, andmethods of operably linking them to a nucleic acid sequence encoding anattenuation protein are known in the art.

In certain embodiments, a recombinant bacterium of the invention maycomprise ΔP_(spoT)::TT araC P_(BAD) spoT or ΔP_(crp)::TT araC P_(BAD)crp, or a combination thereof. (P stands for promoter and TT stands fortranscription terminator). Growth of such strains in the presence ofarabinose leads to transcription of the spoT, and/or crp nucleic acidsequences, but nucleic acid sequence expression ceases in a host becausethere is no free arabinose. Attenuation develops as the products of thespoT, and/or the crp nucleic acid sequences are diluted at each celldivision. Generally speaking, the concentration of arabinose necessaryto induce expression is typically less than about 2%. In someembodiments, the concentration is less than about 1.5%, 1%, 0.5%, 0.2%,0.1%, or 0.05%. In certain embodiments, the concentration may be about0.04%, 0.03%, 0.02%, or 0.01%. In an exemplary embodiment, theconcentration is about 0.05%. Higher concentrations of arabinose orother sugars may lead to acid production during growth that may inhibitdesirable cell densities. The inclusion of mutations such as ΔaraBA ormutations that block the uptake and/or breakdown of maltose, rhamnose,or xylose, however, may prevent such acid production and enable use ofhigher sugar concentrations with no ill effects.

When the regulatable promoter is responsive to arabinose, the onset ofattenuation may be delayed by including additional mutations, such asΔaraBA, which prevents use of arabinose retained in the cell cytoplasmat the time of oral immunization, and/or ΔaraFGH that enhances retentionof arabinose. Thus, inclusion of these mutations may be beneficial in atleast two ways: first, enabling higher culture densities, and secondenabling a further delay in the display of the attenuated phenotype thatmay result in higher densities of the Yersinia vaccine strain ineffector lymphoid tissues to further enhance immunogenicity.

(c) Modifications

Attenuation of the recombinant bacterium may be optimized by modifyingthe nucleic acid sequence encoding an attenuation protein and/orpromoter. Methods of modifying a promoter and/or a nucleic acid sequenceencoding an attenuation protein are the same as those detailed belowwith respect to repressors in Section II.

In some embodiments, more than one modification may be performed tooptimize the attenuation of the bacterium. For instance, at least one,two, three, four, five, six, seven, eight or nine modifications may beperformed to optimize the attenuation of the bacterium.

(d) Crp Cassette

In some embodiments, a recombinant bacterium of the invention may alsocomprise a ΔP_(crp)::TT araC P_(BAD) crp deletion-insertion mutation, asdescribed above. Since the araC P_(BAD) cassette is dependent both onthe presence of arabinose and the binding of the catabolite repressorprotein Crp, a ΔP_(crp)::TT araC P_(BAD) crp deletion-insertion mutationmay be included as an additional control on the expression of thenucleic acid sequence encoding an attenuation protein also controlled byan araC P_(BAD) crp deletion-insertion cassette.

Generally speaking, the activity of the Crp protein requires interactionwith cAMP, but the addition of glucose, which may inhibit synthesis ofcAMP, decreases the ability of the Crp protein to regulate transcriptionfrom the araC P_(BAD) promoter. Consequently, to avoid the effect ofglucose on cAMP, glucose may be substantially excluded from the growthmedia, or variants of crp may be isolated that synthesize a Crp proteinthat is not dependent on cAMP to regulate transcription from P_(BAD).This strategy may also be used in other systems responsive to Crp, suchas the systems responsive to rhamnose and xylose described above

(e) Regulated Expression

In each of the above embodiments, a bacterium capable of regulatedattenuation may also be capable of regulated expression of at least onenucleic acid encoding an antigen as detailed in section II below.

(f) Attenuation

In addition to comprising a regulated attenuation mutation, a bacteriumof the invention may be further attenuated. Other methods of attenuationare known in the art. For instance, attenuation may be accomplished byaltering (e.g., deleting) native nucleic acid sequences found in thewild-type bacterium. For instance, non-limiting examples of nucleic acidsequences which may be used for attenuation may include: a pab nucleicacid sequence, a pur nucleic acid sequence, an aro nucleic acidsequence, asd, a dap nucleic acid sequence, nadA, pncB, gale (lse), pmi,fur, rpsL, ompR, htrA, hemA, cya, crp, dam, phoP, phoQ, rfc, poxA, galU,mviA (hnr), sodC, recA, rpoE, flgM, tonB, slyA, pla, pabA, pabB, pabC,yopH and any combination thereof. Exemplary attenuating mutations may bedesigned in aroA, aroC, aroD, cya, crp, phoP, phoQ, ompR, galE (lse),pabA, pabB, pabC and htrA.

In certain embodiments, a nucleic acid sequence listed above may beplaced under the control of a sugar regulated promoter wherein the sugaris present during in vitro growth of the recombinant bacterium, butsubstantially absent within an animal or human host. The cessation intranscription of a nucleic acid sequence listed above would then resultin attenuation and the inability of the recombinant bacterium to inducedisease symptoms.

In another embodiment, a recombinant bacterium may contain one and insome embodiments, more than one, deletion and/or deletion-insertionmutation present in the strains listed in Table 3. Furthermore, vectors,as listed in Table 3, and described in the Examples below, along withother plasmid vectors, may be used to introduce these deletion anddeletion-insertion mutations into strains during their construction.Methods of introducing these mutations into a strain are known in theart and detailed in the Examples.

The bacterium may also be modified to create a balanced-lethalhost-vector system, although other types of systems may also be used(e.g., creating complementation heterozygotes). For the balanced-lethalhost-vector system, the bacterium may be modified by manipulating itsability to synthesize various essential constituents needed forsynthesis of the rigid peptidoglycan layer of its cell wall. In oneexample, the constituent is diaminopimelic acid (DAP). Various enzymesare involved in the eventual synthesis of DAP. In one example, thebacterium is modified by using a ΔasdA mutation to eliminate thebacterium's ability to produce β-aspartate semialdehyde dehydrogenase,an enzyme essential for the synthesis of DAP. One of skill in the artcan also use the teachings of U.S. Pat. No. 6,872,547 for other types ofmutations of nucleic acid sequences that result in the abolition of thesynthesis of DAP. These nucleic acid sequences may include, but are notlimited to, dapA, dapB, dapC, dapD, dapE, dapF, and asd. Othermodifications that may be employed include modifications to abacterium's ability to synthesize D-alanine or to synthesize D-glutamicacid (e.g., ΔmurI mutations), which are both unique constituents of thepeptidoglycan layer of the Yersinia bacterial cell wall

Yet another balanced-lethal host-vector system comprises modifying thebacterium such that the synthesis of an essential constituent of therigid layer of the bacterial cell wall is dependent on a nutrient (e.g.,arabinose) that can be supplied during the growth of the microorganism.For example, a bacterium may be comprise the ΔP_(murA)::TT araC P_(BAD)murA deletion-insertion mutation. This type of mutation makes synthesisof muramic acid (another unique essential constituent of thepeptidoglycan layer of the bacterial cell wall) dependent on thepresence of arabinose that can be supplied during growth of thebacterium in vitro.

When arabinose is absent, however, as it is in an animal or human host,the essential constituent of the peptidoglycan layer of the cell wall isnot synthesized. This mutation represents an arabinose-dependant lethalmutation. In the absence of arabinose, synthesis of muramic acid ceasesand lysis of the bacterium occurs because the peptidoglycan layer of thecell wall is not synthesized. It is not possible to generate ΔmurAmutations because they are lethal. The necessary nutrient, aphosphorylated muramic acid, cannot be exogenously supplied becauseenteric bacteria cannot take the nutrient up from the media. Recombinantbacteria with a ΔP_(murA)::TT araC P_(BAD) murA deletion-insertionmutation grown in the presence of arabinose exhibit effectivecolonization of effector lymphoid tissues after oral vaccination priorto undergoing lysis due to the inability to synthesize muramic acid.

Similarly, various embodiments may comprise the araC P_(BAD) c2 cassetteinserted into the asd nucleic acid sequence that encodes aspartatesemialdehyde dehydrogenase. Since the araC nucleic acid sequence istranscribed in a direction that could lead to interference in theexpression of adjacent nucleic acid sequences and adversely affectvaccine strain performance, a transcription termination (TT) sequence isgenerally inserted 3′ to the araC nucleic acid sequence. The chromosomalasd nucleic acid sequence is typically inactivated to enable use ofplasmid vectors encoding the wild-type asd nucleic acid sequence in thebalanced-lethal host-vector system. This allows stable maintenance ofplasmids in vivo in the absence of any drug resistance attributes thatare not permissible in live bacterial vaccines. In some of theseembodiments, the wild-type asd nucleic acid sequence may be encoded bythe vector described above. The vector enables the regulated expressionof an antigen encoding sequence through the repressible promoter.

In further embodiments, the bacterium may be attenuated by regulatingthe murA nucleic acid sequence encoding the first enzyme in muramic acidsynthesis and the asd nucleic acid sequence essential for DAP synthesis.This host-vector grows in LB broth with 0.1% L-arabinose, but is unableto grow in or on media devoid of arabinose since it undergoes cellwall-less death by lysis. In some embodiments of the invention, therecombinant bacterium may comprise araBA and araFGH mutations topreclude breakdown and leakage of internalized arabinose such that asdand murA nucleic acid sequence expression continues for a cell divisionor two after oral immunization into an environment that is devoid ofexternal arabinose.

II. Regulated Expression

The present invention encompasses a recombinant bacterium capable of theregulated expression of at least one nucleic acid sequence encoding anantigen of interest. The regulated expression may allow, in certainembodiments, the recombinant bacterium to elicit both a humoral and acellular immune response to the antigen.

Generally speaking, the bacterium comprises a chromosomally integratednucleic acid sequence encoding a repressor and a vector. Each isdiscussed in more detail below.

(a) Chromosomally Integrated Nucleic Acid Sequence Encoding a Repressor

A recombinant bacterium of the invention that is capable of theregulated expression of at least one nucleic acid sequence encoding anantigen comprises, in part, at least one chromosomally integratednucleic acid sequence encoding a repressor. Typically, the nucleic acidsequence encoding a repressor is operably linked to a regulatablepromoter. The nucleic acid sequence encoding a repressor and/or thepromoter may be modified from the wild-type nucleic acid sequence so asto optimize the expression level of the nucleic acid sequence encodingthe repressor.

Methods of chromosomally integrating a nucleic acid sequence encoding arepressor operably-linked to a regulatable promoter are known in the artand detailed in the examples. Generally speaking, the nucleic acidsequence encoding a repressor should not be integrated into a locus thatdisrupts colonization of the host by the recombinant bacterium, orattenuates the bacterium.

In some embodiments, at least one nucleic acid sequence encoding arepressor is chromosomally integrated. In other embodiments, at leasttwo, or at least three nucleic acid sequences encoding repressors may bechromosomally integrated into the recombinant bacterium. If there ismore than one nucleic acid sequence encoding a repressor, each nucleicacid sequence encoding a repressor may be operably linked to aregulatable promoter, such that each promoter is regulated by the samecompound or condition. Alternatively, each nucleic acid sequenceencoding a repressor may be operably linked to a regulatable promoter,each of which is regulated by a different compound or condition.

i. Repressor

As used herein, “repressor” refers to a biomolecule that repressestranscription from one or more promoters. Generally speaking, a suitablerepressor of the invention is synthesized in high enough quantitiesduring the in vitro growth of the bacterial strain to repress thetranscription of the nucleic acid encoding an antigen of interest on thevector, as detailed below, and not impede the in vitro growth of thestrain. Additionally, a suitable repressor will generally besubstantially stable, i.e. not subject to proteolytic breakdown.Furthermore, a suitable repressor will be diluted by about half at everycell division after expression of the repressor ceases, such as in anon-permissive environment (e.g. an animal or human host).

The choice of a repressor depends, in part, on the species of therecombinant bacterium used. For instance, the repressor is usually notderived from the same species of bacteria as the recombinant bacterium.For instance, the repressor may be derived from E. coli. Alternatively,the repressor may be from a bacteriophage.

Suitable repressors are known in the art, and may include, for instance,LacI of E. coli, C2 encoded by bacteriophage P22, or C1 encoded bybacteriophage λ. Other suitable repressors may be repressors known toregulate the expression of a regulatable nucleic acid sequence, such asnucleic acid sequences involved in the uptake and utilization of sugars.In one embodiment, the repressor is LacI. In another embodiment, therepressor is C2. In yet another embodiment, the repressor is C1.

ii. Regulatable Promoter

The chromosomally integrated nucleic acid sequence encoding a repressoris operably linked to a regulatable promoter. The term “promoter”, asused herein, may mean a synthetic or naturally-derived molecule that iscapable of conferring, activating or enhancing expression of a nucleicacid. A promoter may comprise one or more specific transcriptionalregulatory sequences to further enhance expression and/or to alter thespatial expression and/or temporal expression of a nucleic acid. Theterm “operably linked,” is defined above.

The regulated promoter used herein generally allows transcription of thenucleic acid sequence encoding a repressor while in a permissiveenvironment (i.e. in vitro growth), but ceases transcription of thenucleic acid sequence encoding a repressor while in a non-permissiveenvironment (i.e. during growth of the bacterium in an animal or humanhost). For instance, the promoter may be sensitive to a physical orchemical difference between the permissive and non-permissiveenvironment. Suitable examples of such regulatable promoters are knownin the art.

In some embodiments, the promoter may be responsive to the level ofarabinose in the environment. Generally speaking, arabinose may bepresent during the in vitro growth of a bacterium, while typicallyabsent from host tissue. In one embodiment, the promoter is derived froman araC-P_(BAD) system. The araC-P_(BAD) system is a tightly regulatedexpression system that has been shown to work as a strong promoterinduced by the addition of low levels of arabinose. The araC-araBADpromoter is a bidirectional promoter controlling expression of thearaBAD nucleic acid sequences in one direction, and the araC nucleicacid sequence in the other direction. For convenience, the portion ofthe araC-araBAD promoter that mediates expression of the araBAD nucleicacid sequences, and which is controlled by the araC nucleic acidsequence product, is referred to herein as P_(BAD). For use as describedherein, a cassette with the araC nucleic acid sequence and thearaC-araBAD promoter may be used. This cassette is referred to herein asaraC-P_(BAD). The AraC protein is both a positive and negative regulatorof P_(BAD). In the presence of arabinose, the AraC protein is a positiveregulatory element (i.e., is an activator) that allows expression fromP_(BAD). In the absence of arabinose, the AraC protein repressesexpression from P_(BAD). This can lead to a 1,200-fold difference in thelevel of expression from P_(BAD) (i.e., is a repressor).

Other enteric bacteria contain arabinose regulatory systems homologousto the araC araBAD system from E. coli. For example, there is homologyat the amino acid sequence level between the E. coli and the S.Typhimurium AraC proteins, and less homology at the DNA level. However,there is high specificity in the activity of the AraC proteins. Forexample, the E. coli AraC protein activates only E. coli P_(BAD) (in thepresence of arabinose) and not S. Typhimurium P_(BAD). Thus, anarabinose-regulated promoter may be used in a recombinant bacterium thatpossesses a similar arabinose operon, without substantial interferencebetween the two, if the promoter and the operon are derived from twodifferent species of bacteria.

Generally speaking, the concentration of arabinose necessary to induceexpression is typically less than about 2%. In some embodiments, theconcentration is less than about 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05%.In other embodiments, the concentration is 0.05% or below, e.g. about0.04%, 0.03%, 0.02%, or 0.01%. In an exemplary embodiment, theconcentration is about 0.05%.

In other embodiments, the promoter may be responsive to the level ofmaltose in the environment. Generally speaking, maltose may be presentduring the in vitro growth of a bacterium, while typically absent fromhost tissue. The malT nucleic acid encodes MalT, a positive regulator offour maltose-responsive promoters (P_(PQ), P_(EFG), P_(KBM), and P_(S)).The combination of malT and a mal promoter creates a tightly regulatedexpression system that has been shown to work as a strong promoterinduced by the addition of maltose. Unlike the araC-P_(BAD) system, malTis expressed from a promoter (P_(T)) functionally unconnected to theother mal promoters. P_(T) is not regulated by MalT. The malEFG-malKBMpromoter is a bidirectional promoter controlling expression of themalKBM nucleic acid sequences in one direction, and the malEFG nucleicacid sequences in the other direction. For convenience, the portion ofthe malEFG-malKBM promoter that mediates expression of the malKBMnucleic acid sequence, and which is controlled by the malT nucleic acidsequence product, is referred to herein as P_(KBM), and the portion ofthe malEFG-malKBM promoter that mediates expression of the malEFGnucleic acid sequence, and that is controlled by the malT nucleic acidsequence product, is referred to herein as P_(EFG). Full induction ofP_(KBM) requires the presence of the MalT binding sites of P_(EFG). Foruse in the vectors and systems described herein, a cassette with themalT nucleic acid sequence and one of the mal promoters may be used.This cassette is referred to herein as malT-P_(mal). In the presence ofmaltose, the MalT protein is a positive regulatory element that allowsexpression from P_(mal).

In still other embodiments, the promoter may be sensitive to the levelof rhamnose in the environment. Analogous to the araC-P_(BAD) systemdescribed above, the rhaRS-P_(rhaB) activator-promoter system is tightlyregulated by rhamnose. Expression from the rhamnose promoter (P_(rha))is induced to high levels by the addition of rhamnose, which is commonin bacteria but rarely found in host tissues. The nucleic acid sequencesrhaBAD are organized in one operon that is controlled by the P_(rhaBAD)promoter. This promoter is regulated by two activators, RhaS and RhaR,and the corresponding nucleic acid sequences belong to one transcriptionunit that is located in the opposite direction of the rhaBAD nucleicacid sequences. If L-rhamnose is available, RhaR binds to the P_(rhaRS)promoter and activates the production of RhaR and RhaS. RhaS togetherwith L-rhamnose in turn binds to the P_(rhaBAD) and the P_(rhaT)promoter and activates the transcription of the structural nucleic acidsequences. Full induction of rhaBAD transcription also requires bindingof the Crp-cAMP complex, which is a key regulator of cataboliterepression.

Although both L-arabinose and L-rhamnose act directly as inducers forexpression of regulons for their catabolism, important differences existin regard to the regulatory mechanisms. L-Arabinose acts as an inducerwith the activator AraC in the positive control of the arabinoseregulon. However, the L-rhamnose regulon is subject to a regulatorycascade; it is therefore subject to even tighter control than the araCP_(BAD) system. L-Rhamnose acts as an inducer with the activator RhaRfor synthesis of RhaS, which in turn acts as an activator in thepositive control of the rhamnose regulon. In the present invention,rhamnose may be used to interact with the RhaR protein and then the RhaSprotein may activate transcription of a nucleic acid sequenceoperably-linked to the P_(rhaBAD) promoter.

In still other embodiments, the promoter may be sensitive to the levelof xylose in the environment. The xylR-P_(xylA) system is anotherwell-established inducible activator-promoter system. Xylose inducesxylose-specific operons (xylE, xylFGHR, and xylAB) regulated by XylR andthe cyclic AMP-Crp system. The XylR protein serves as a positiveregulator by binding to two distinct regions of the xyl nucleic acidsequence promoters. As with the araC-P_(BAD) system described above, thexylR-P_(xylAB) and/or xylR-P_(xylFGH) regulatory systems may be used inthe present invention. In these embodiments, xylR P_(xylAB) xyloseinteracting with the XylR protein activates transcription of nucleicacid sequences operably-linked to either of the two P_(xyl) promoters.

The nucleic acid sequences of the promoters detailed herein are known inthe art, and methods of operably-linking them to a chromosomallyintegrated nucleic acid sequence encoding a repressor are known in theart and detailed in the examples.

iii. Modification to Optimize Expression

A nucleic acid sequence encoding a repressor and regulatable promoterdetailed above, for use in the present invention, may be modified so asto optimize the expression level of the nucleic acid sequence encodingthe repressor. The optimal level of expression of the nucleic acidsequence encoding the repressor may be estimated, or may be determinedby experimentation (see the Examples). Such a determination should takeinto consideration whether the repressor acts as a monomer, dimer,trimer, tetramer, or higher multiple, and should also take intoconsideration the copy number of the vector encoding the antigen ofinterest, as detailed below. In an exemplary embodiment, the level ofexpression is optimized so that the repressor is synthesized while inthe permissive environment (i.e. in vitro growth) at a level thatsubstantially inhibits the expression of the nucleic acid encoding anantigen of interest, and is substantially not synthesized in anon-permissive environment, thereby allowing expression of the nucleicacid encoding an antigen of interest.

As stated above, the level of expression may be optimized by modifyingthe nucleic acid sequence encoding the activator, repressor and/orpromoter. As used herein, “modify” refers to an alteration of thenucleic acid sequence of the repressor and/or promoter that results in achange in the level of transcription of the nucleic acid sequenceencoding the repressor, or that results in a change in the level ofsynthesis of the repressor. For instance, in one embodiment, modify mayrefer to altering the start codon of the nucleic acid sequence encodingthe repressor. Generally speaking, a GTG or TTG start codon, as opposedto an ATG start codon, may decrease translation efficiency ten-fold. Inanother embodiment, modify may refer to altering the Shine-Dalgarno (SD)sequence of the nucleic acid sequence encoding the repressor. The SDsequence is a ribosomal binding site generally located 6-7 nucleotidesupstream of the start codon. The SD consensus sequence is AGGAGG, andvariations of the consensus sequence may alter translation efficiency.In yet another embodiment, modify may refer to altering the distancebetween the SD sequence and the start codon. In still anotherembodiment, modify may refer to altering the −35 sequence for RNApolymerase recognition. In a similar embodiment, modify may refer toaltering the −10 sequence for RNA polymerase binding. In an additionalembodiment, modify may refer to altering the number of nucleotidesbetween the −35 and −10 sequences. In an alternative embodiment, modifymay refer to optimizing the codons of the nucleic acid sequence encodingthe repressor to alter the level of translation of the mRNA encoding therepressor. For instance, non-A rich codons initially after the startcodon of the nucleic acid sequence encoding the repressor may notmaximize translation of the mRNA encoding the repressor. Similarly, thecodons of the nucleic acid sequence encoding the repressor may bealtered so as to mimic the codons from highly synthesized proteins of aparticular organism. In a further embodiment, modify may refer toaltering the GC content of the nucleic acid sequence encoding therepressor to change the level of translation of the mRNA encoding therepressor.

In some embodiments, more than one modification or type of modificationmay be performed to optimize the expression level of the nucleic acidsequence encoding the repressor. For instance, at least one, two, three,four, five, six, seven, eight, or nine modifications, or types ofmodifications, may be performed to optimize the expression level of thenucleic acid sequence encoding the repressor.

Methods of modifying the nucleic acid sequence encoding the repressorand/or the regulatable promoter are known in the art and detailed in theexamples.

iv. Transcription Termination Sequence

In some embodiments, the chromosomally integrated nucleic acid sequenceencoding the repressor further comprises a transcription terminationsequence. A transcription termination sequence may be included toprevent inappropriate expression of nucleic acid sequences adjacent tothe chromosomally integrated nucleic acid sequence encoding therepressor or activator and regulatable promoter.

(b) Vector

A recombinant bacterium of the invention that is capable of theregulated expression of at least one nucleic acid sequence encoding anantigen comprises, in part, a vector. The vector comprises a nucleicacid sequence encoding at least one antigen of interest operably linkedto a promoter. The promoter is regulated by the chromosomally encodedrepressor, such that the expression of the nucleic acid sequenceencoding an antigen is repressed during in vitro growth of thebacterium, but the bacterium is capable of high level synthesis of theantigen in an animal or human host.

As used herein, “vector” refers to an autonomously replicating nucleicacid unit. The present invention can be practiced with any known type ofvector, including viral, cosmid, phasmid, and plasmid vectors. The mostpreferred type of vector is a plasmid vector.

As is well known in the art, plasmids and other vectors may possess awide array of promoters, multiple cloning sequences, transcriptionterminators, etc., and vectors may be selected so as to control thelevel of expression of the nucleic acid sequence encoding an antigen bycontrolling the relative copy number of the vector. In some instances inwhich the vector might encode a surface localized adhesin as theantigen, or an antigen capable of stimulating T-cell immunity, it may bepreferable to use a vector with a low copy number such as at least two,three, four, five, six, seven, eight, nine, or ten copies per bacterialcell. A non-limiting example of a low copy number vector may be a vectorcomprising the pSC101 ori.

In other cases, an intermediate copy number vector might be optimal forinducing desired immune responses. For instance, an intermediate copynumber vector may have at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 copies per bacterial cell.A non-limiting example of an intermediate copy number vector may be avector comprising the p15A ori.

In still other cases, a high copy number vector might be optimal for theinduction of maximal antibody responses. A high copy number vector mayhave at least 31, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100 copies per bacterial cell. In some embodiments, a high copy numbervector may have at least 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, or 400 copies per bacterial cell. Non-limiting examplesof high copy number vectors may include a vector comprising the pBR orior the pUC ori.

Additionally, vector copy number may be increased by selecting formutations that increase plasmid copy number. These mutations may occurin the bacterial chromosome but are more likely to occur in the plasmidvector.

Preferably, vectors used herein do not comprise antibiotic resistancemarkers to select for maintenance of the vector.

i. Antigen

As used herein, “antigen” refers to a biomolecule capable of elicitingan immune response in a host. In some embodiments, an antigen may be aprotein, or fragment of a protein, or a nucleic acid. In an exemplaryembodiment, the antigen elicits a protective immune response. As usedherein, “protective” means that the immune response contributes to thelessening of any symptoms associated with infection of a host with thepathogen the antigen was derived from or designed to elicit a responseagainst. For example, a protective antigen from a pathogen, such asMycobacterium, may induce an immune response that helps to amelioratesymptoms associated with Mycobacterium infection or reduce the morbidityand mortality associated with infection with the pathogen. The use ofthe term “protective” in this invention does not necessarily requirethat the host is completely protected from the effects of the pathogen.

Antigens may be from bacterial, viral, mycotic and parasitic pathogens,and may be designed to protect against bacterial, viral, mycotic, andparasitic infections, respectively. Alternatively, antigens may bederived from gametes, provided they are gamete specific, and may bedesigned to block fertilization. In another alternative, antigens may betumor antigens, and may be designed to decrease tumor growth. It isspecifically contemplated that antigens from organisms newly identifiedor newly associated with a disease or pathogenic condition, or new oremerging pathogens of animals or humans, including those now known oridentified in the future, may be expressed by a bacterium detailedherein. Furthermore, antigens for use in the invention are not limitedto those from pathogenic organisms. Immunogenicity of the bacterium maybe augmented and/or modulated by constructing strains that also expresssequences for cytokines, adjuvants, and other immunomodulators.

Some examples of microorganisms useful as a source for antigen arelisted below. These may include microoganisms for the control of plaguecaused by Yersinia pestis and other Yersinia species such as Y.pseudotuberculosis and Y. enterocolitica, for the control of gonorrheacaused by Neisseria gonorrhoea, for the control of syphilis caused byTreponema pallidum, and for the control of venereal diseases as well aseye infections caused by Chlamydia trachomatis. Species of Streptococcusfrom both group A and group B, such as those species that cause sorethroat or heart diseases, Erysipelothrix rhusiopathiae, Neisseriameningitidis, Mycoplasma pneumoniae and other Mycoplasma-species,Hemophilus influenza, Bordetella pertussis, Mycobacterium tuberculosis,Mycobacterium leprae, other Bordetella species, Escherichia coli,Streptococcus equi, Streptococcus pneumoniae, Brucella abortus,Pasteurella hemolytica and P. multocida, Vibrio cholera, Shigellaspecies, Borrellia species, Bartonella species, Heliobacter pylori,Campylobacter species, Pseudomonas species, Moraxella species, Brucellaspecies, Francisella species, Aeromonas species, Actinobacillus species,Clostridium species, Rickettsia species, Bacillus species, Coxiellaspecies, Ehrlichia species, Listeria species, and Legionella pneumophilaare additional examples of bacteria within the scope of this inventionfrom which antigen nucleic acid sequences could be obtained. Viralantigens may also be used. Viral antigens may be used in antigendelivery microorganisms directed against viruses, either DNA or RNAviruses, for example from the classes Papovavirus, Adenovirus,Herpesvirus, Poxvirus, Parvovirus, Reovirus, Picornavirus, Myxovirus,Paramyxovirus, Flavivirus or Retrovirus. Antigens may also be derivedfrom pathogenic fungi, protozoa and parasites.

Certain embodiments encompass an allergen as an antigen. Allergens aresubstances that cause allergic reactions in a host that is exposed tothem. Allergic reactions, also known as Type I hypersensitivity orimmediate hypersensitivity, are vertebrate immune responsescharacterized by IgE production in conjunction with certain cellularimmune reactions. Many different materials may be allergens, such asanimal dander and pollen, and the allergic reaction of individual hostswill vary for any particular allergen. It is possible to inducetolerance to an allergen in a host that normally shows an allergicresponse. The methods of inducing tolerance are well-known and generallycomprise administering the allergen to the host in increasing dosages.

It is not necessary that the vector comprise the complete nucleic acidsequence of the antigen. It is only necessary that the antigen sequenceused be capable of eliciting an immune response. The antigen may be onethat was not found in that exact form in the parent organism. Forexample, a sequence coding for an antigen comprising 100 amino acidresidues may be transferred in part into a recombinant bacterium so thata peptide comprising only 75, 65, 55, 45, 35, 25, 15, or even 10, aminoacid residues is produced by the recombinant bacterium. Alternatively,if the amino acid sequence of a particular antigen or fragment thereofis known, it may be possible to chemically synthesize the nucleic acidfragment or analog thereof by means of automated nucleic acid sequencesynthesizers, PCR, or the like and introduce said nucleic acid sequenceinto the appropriate copy number vector.

In another alternative, a vector may comprise a long sequence of nucleicacid encoding several nucleic acid sequence products, one or all ofwhich may be antigenic. In some embodiments, a vector of the inventionmay comprise a nucleic acid sequence encoding at least one antigen, atleast two antigens, at least three antigens, or more than threeantigens. These antigens may be encoded by two or more open readingframes operably linked to be expressed coordinately as an operon,wherein each antigen is synthesized independently. Alternatively, thetwo or more antigens may be encoded by a single open reading frame suchthat the antigens are synthesized as a fusion protein.

In certain embodiments, an antigen of the invention may comprise a Bcell epitope or a T cell epitope. Alternatively, an antigen to which animmune response is desired may be expressed as a fusion to a carrierprotein that contains a strong promiscuous T cell epitope and/or servesas an adjuvant and/or facilitates presentation of the antigen toenhance, in all cases, the immune response to the antigen or itscomponent part. This can be accomplished by methods known in the art.Fusion to tenus toxin fragment C, CT-B, LT-B and hepatitis virus B coreare particularly useful for these purposes, although other epitopepresentation systems are well known in the art.

In further embodiments, a nucleic acid sequence encoding an antigen ofthe invention may comprise a secretion signal. In other embodiments, anantigen of the invention may be toxic to the recombinant bacterium.

A suitable antigen derived from Yersinia, and designed to induce animmune response against Yersinia may include LcrV, Psn, PsaA, and Pla.

ii. Promoter Regulated by Repressor

The vector comprises a nucleic acid sequence encoding at least oneantigen operably-linked to a promoter regulated by the repressor,encoded by a chromosomally integrated nucleic acid sequence. One ofskill in the art would recognize, therefore, that the selection of arepressor dictates, in part, the selection of the promoteroperably-linked to a nucleic acid sequence encoding an antigen ofinterest. For instance, if the repressor is LacI, then the promoter maybe selected from the group consisting of LacI responsive promoters, suchas P_(trc), P_(lac), P_(T7lac) and P_(tac). If the repressor is C2, thenthe promoter may be selected from the group consisting of C2 responsivepromoters, such as P22 promoters P_(L) and P_(R). If the repressor isC1, then the promoter may be selected from the group consisting of C1responsive promoters, such as X promoters P_(L) and P_(R).

In each embodiment herein, the promoter regulates expression of anucleic acid sequence encoding the antigen, such that expression of thenucleic acid sequence encoding an antigen is repressed when therepressor is synthesized (i.e. during in vitro growth of the bacterium),but expression of the nucleic acid sequence encoding an antigen is highwhen the repressor is not synthesized (i.e. in an animal or human host).Generally speaking, the concentration of the repressor will decreasewith every cell division after expression of the nucleic acid sequenceencoding the repressor ceases. In some embodiments, the concentration ofthe repressor decreases enough to allow high-level expression of thenucleic acid sequence encoding an antigen after about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 divisions of the bacterium. In an exemplaryembodiment, the concentration of the repressor decreases enough to allowhigh level expression of the nucleic acid sequence encoding an antigenafter about 5 divisions of the bacterium in an animal or human host.

In certain embodiments, the promoter may comprise other regulatoryelements. For instance, the promoter may comprise lacO if the repressoris LacI. This is the case with the lipoprotein promoter P_(lpp) that isregulated by LacI since it possesses the LacI binding domain lacO.

In one embodiment, the repressor is a LacI repressor and the promoter isP_(trc).

iii. Expression of the Nucleic Acid Sequence Encoding an Antigen

As detailed above, generally speaking the expression of the nucleic acidsequence encoding the antigen should be repressed when the repressor issynthesized. For instance, if the repressor is synthesized during invitro growth of the bacterium, expression of the nucleic acid sequenceencoding the antigen should be repressed. Expression may be “repressed”or “partially repressed” when it is about 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, 1%, or even less than 1% of the expression undernon-repressed conditions. Thus although the level of expression underconditions of “complete repression” might be exceeding low, it is likelyto be detectable using very sensitive methods since repression can neverby absolute.

Conversely, the expression of the nucleic acid sequence encoding theantigen should be high when the expression of the nucleic acid sequenceencoding the repressor is repressed. For instance, if the nucleic acidsequence encoding the repressor is not expressed during growth of therecombinant bacterium in the host, the expression of the nucleic acidsequence encoding the antigen should be high. As used herein, “highlevel” expression refers to expression that is strong enough to elicitan immune response to the antigen. Consequently, the copy numbercorrelating with high level expression can and will vary depending onthe antigen and the type of immune response desired. Methods ofdetermining whether an antigen elicits an immune response such as bymeasuring antibody levels or antigen-dependant T cell populations orantigen-dependant cytokine levels are known in the art, and methods ofmeasuring levels of expression of antigen encoding sequences bymeasuring levels of mRNA transcribed or by quantitating the level ofantigen synthesis are also known in the art. For more details, see theexamples.

(c) Crp Cassette

In some embodiments, a recombinant bacterium of the invention may alsocomprise a ΔP_(crp)::TT araC P_(BAD) crp deletion-insertion mutation.Since the araC P_(BAD) cassette is dependent both on the presence ofarabinose and the binding of the catabolite repressor protein Crp, aΔP_(crp)::TT araC P_(BAD) crp deletion-insertion mutation may beincluded as an additional means to reduce expression of any nucleic acidsequence under the control of the P_(BAD) promoter. This means that whenthe bacterium is grown in a non-permissive environment (i.e. noarabinose) both the repressor itself and the Crp protein cease to besynthesized, consequently eliminating both regulating signals for thearaC P_(BAD) regulated nucleic acid sequence. This double shut off ofaraC P_(BAD) may constitute an additional safety feature ensuring thegenetic stability of the desired phenotypes.

Generally speaking, the activity of the Crp protein requires interactionwith cAMP, but the addition of glucose, which may inhibit synthesis ofcAMP, decreases the ability of the Crp protein to regulate transcriptionfrom the araC P_(BAD) promoter. Consequently, to avoid the effect ofglucose on cAMP, glucose may be substantially excluded from the growthmedia, or variants of crp may be isolated that synthesize a Crp proteinthat is not dependent on cAMP to regulate transcription from P_(BAD).This strategy may also be used in other systems responsive to Crp, suchas the systems responsive to rhamnose and xylose described above.

III. Vaccine Compositions and Administration

A recombinant bacterium of the invention may be administered to a hostas a vaccine composition. As used herein, a vaccine composition may be acomposition designed to elicit an immune response against Yersinia.Additionally, a vaccine composition may be a composition designed toelicit an immune response against Yersinia and against one or moreadditional pathogens. In an exemplary embodiment, the immune response isprotective, as described above. In one exemplary embodiment, the immuneresponse is protective against both pneumonic and bubonic plague. Immuneresponses to antigens are well studied and widely reported. A survey ofimmunology is given by Paul, W E, Stites D et. al. and Ogra P L. et. al.Mucosal immunity is also described by Ogra P L et. al.

Vaccine compositions of the present invention may be administered to anyhost capable of mounting an immune response. Such hosts may include allvertebrates, for example, mammals, including domestic animals,agricultural animals, laboratory animals, and humans. Preferably, thehost is a warm-blooded animal. The vaccine can be administered as aprophylactic, for treatment purposes, or for possible elimination of Y.pestis persistence in wild-animals.

In exemplary embodiments, the recombinant bacterium is alive whenadministered to a host in a vaccine composition of the invention.Suitable vaccine composition formulations and methods of administrationare detailed below.

(a) Vaccine Composition

A vaccine composition comprising a recombinant bacterium of theinvention may optionally comprise one or more possible additives, suchas carriers, preservatives, stabilizers, adjuvants, and othersubstances.

In one embodiment, the vaccine comprises an adjuvant. Adjuvants, such asaluminum hydroxide or aluminum phosphate, are optionally added toincrease the ability of the vaccine to trigger, enhance, or prolong animmune response. In exemplary embodiments, the use of a live attenuatedrecombinant bacterium may act as a natural adjuvant. The vaccinecompositions may further comprise additional components known in the artto improve the immune response to a vaccine, such as T cellco-stimulatory molecules or antibodies, such as anti-CTLA4. Additionalmaterials, such as cytokines, chemokines, and bacterial nucleic acidsequences naturally found in bacteria, like CpG, are also potentialvaccine adjuvants.

In another embodiment, the vaccine may comprise a pharmaceutical carrier(or excipient). Such a carrier may be any solvent or solid material forencapsulation that is non-toxic to the inoculated host and compatiblewith the recombinant bacterium. A carrier may give form or consistency,or act as a diluent. Suitable pharmaceutical carriers may include liquidcarriers, such as normal saline and other non-toxic salts at or nearphysiological concentrations, and solid carriers not used for humans,such as talc or sucrose, or animal feed. Carriers may also includestabilizing agents, wetting and emulsifying agents, salts for varyingosmolarity, encapsulating agents, buffers, and skin penetrationenhancers. Carriers and excipients as well as formulations forparenteral and nonparenteral drug delivery are set forth in Remington'sPharmaceutical Sciences 19th Ed. Mack Publishing (1995). When used foradministering via the bronchial tubes, the vaccine is preferablypresented in the form of an aerosol.

Care should be taken when using additives so that the live recombinantbacterium is not killed, or have its ability to effectively colonize thehost compromised by the use of additives. Stabilizers, such as lactoseor monosodium glutamate (MSG), may be added to stabilize the vaccineformulation against a variety of conditions, such as temperaturevariations or a freeze-drying process.

The dosages of a vaccine composition of the invention can and will varydepending on the recombinant bacterium, the regulated antigen, and theintended host, as will be appreciated by one of skill in the art.Generally speaking, the dosage need only be sufficient to elicit aprotective immune response in a majority of hosts. Routineexperimentation may readily establish the required dosage. Typicalinitial dosages of vaccine for oral administration could be about 1×10⁷to 1×10¹⁰ CFU depending upon the age of the host to be immunized.Administering multiple dosages may also be used as needed to provide thedesired level of protective immunity.

(b) Methods of Administration

A vaccine of the invention may be administered via any suitable route,such as by oral administration, intranasal administration, gastricintubation or in the form of aerosols. Additionally, other methods ofadministering the recombinant bacterium, such as intravenous,intramuscular, subcutaneous injection or other parenteral routes, arepossible.

In some embodiments, these compositions are formulated foradministration by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.). Accordingly, these compositionsare preferably combined with pharmaceutically acceptable vehicles suchas saline, Ringer's solution, dextrose solution, and the like.

IV. Kits

The invention also encompasses kits comprising any one of thecompositions above in a suitable aliquot for vaccinating a host in needthereof. In one embodiment, the kit further comprises instructions foruse. In other embodiments, the composition is lyophilized such thataddition of a hydrating agent (e.g., buffered saline) reconstitutes thecomposition to generate a vaccine composition ready to administer,preferably orally.

V. Methods of Use

A further aspect of the invention encompasses methods of using arecombinant bacterium of the invention. For instance, in one embodimentthe invention provides a method for modulating a host's immune system.The method comprises administering to the host an effective amount of acomposition comprising a recombinant bacterium of the invention. One ofskill in the art will appreciate that an effective amount of acomposition is an amount that will generate the desired immune response(e.g., mucosal, humoral or cellular). Methods of monitoring a host'simmune response are well-known to physicians and other skilledpractitioners. For instance, assays such as ELISA, and ELISPOT may beused. Effectiveness may be determined by monitoring the amount of theantigen of interest remaining in the host, or by measuring a decrease indisease incidence caused by Yersinia and/or another pathogen in a host.For certain pathogens, cultures or swabs taken as biological samplesfrom a host may be used to monitor the existence or amount of pathogenin the individual.

In another embodiment, the invention provides a method for eliciting animmune response against Yersinia in a host. The method comprisesadministering to the host an effective amount of a compositioncomprising a recombinant bacterium of the invention

In still another embodiment, a recombinant bacterium of the inventionmay be used in a method for eliciting an immune response againstYersinia and one or more additional pathogens in an individual in needthereof. The method comprises administrating to the host an effectiveamount of a composition comprising a recombinant bacterium as describedherein.

In a further embodiment, a recombinant bacterium described herein may beused in a method for ameliorating one or more symptoms of bubonic orpneumonic plague in a host in need thereof. The method comprisesadministering an effective amount of a composition comprising arecombinant bacterium as described herein.

EXAMPLES

The following examples illustrate various iterations of the invention.

Introduction for Examples 1-7

The Examples below determine what role relA and spoT play in Y. pestisphysiology and virulence by constructing ΔrelA and ΔrelA ΔspoT mutantsand characterizing them for both in vitro and in vivo characteristics.We examined the effect of these mutations on transcription and proteinlevels at 26° C. (flea temperature) and at 37° C. (human temperature)and the effect on host colonization, immune responses and virulence. Wealso evaluated the double mutant for its capacity to induce protectiveimmunity.

Example 1 Sequence Analysis of the RelA and SpoT Genes

Analysis of the Y. pestis KIM5+ database revealed the presence of relAand spoT genes homologous to E. coli K-12 and S. Typhimurium LT-2 [15,16, 17]. The Y. pestis RelA protein shares 84.7% identity with E. coliK-12 and 83.9% identity with S. Typhimurium LT-2 RelA proteins. The Y.pestis SpoT protein has 91.3% identity with E. coli K-12 and 91.8%identity with S. Typhimurium LT-2 SpoT proteins.

Our analysis indicated that Y. pestis SpoT, but not RelA, possesses theHD domain that is conserved in a superfamily of metal-dependentphosphohydrolases [18]. Histidine (H) and aspartate (D) residues in theHD domain are thought to be involved in (p)ppGpp degradation [18]. BothY. pestis RelA and SpoT proteins possess the conserved ATP/GTP-bindingand GTP binding domains, TGS [19] and ACT [20,21], respectively, thatare present in the E. coli RelA and SpoT proteins [22]. The presence ofthese conserved motifs in the Y. pestis proteins is in agreement withtheir biochemical functions because ATP and GTP are substrates of thereaction catalyzed by (p)ppGpp synthetase.

Example 2 The RelA and SpoT Genes are Involved in Synthesis of ppGpp andPhysiological Differentiation

To evaluate the linkage between relA and spoT and the production ofppGpp, we constructed ΔrelA, ΔrelA ΔspoT [5] and ΔrelA ΔspoT ΔlacZ::TTaraC P_(BAD) spoT mutants of Y. pestis KIM6+ strain (FIG. 1). Toconstruct a strain with arabinose-regulated spoT expression, a TT araCP_(BAD) promoter cassette was inserted in front of the spoT gene. ThespoT gene is located in the middle of an operon. To avoid affecting thetranscription of nearby genes, the TT araC P_(BA)D spoT construct wasinserted at another location, lacZ (FIG. 1).

Because of the high degree of similarity between Y. pestis RelA and SpoTproteins and their E. coli and Salmonella counterparts, it is likelythat the function of RelA and SpoT in Y. pestis will be the same. Toevaluate the effect of relA and spoT on ppGpp synthesis during aminoacid starvation, Y. pestis was grown in PHM2 media [2] withoutL-phenylalanine. ppGpp accumulation was observed in wild-type Y. pestis,but not in the relA null strains (FIG. 2A), illustrating that Y. pestisis indeed capable of ppGpp biosynthesis in response to amino acidstarvation. We also evaluated the effect of carbon starvation. Whenglucose was exhausted in the medium, ppGpp accumulated in the wild typeand ΔrelA spoT+ strains, but not in ΔrelA ΔspoT strains (FIG. 2B). Theseresults indicate that Y. pestis has a RelA-dependent response to aminoacid starvation and a SpoT-dependent response to glucose starvation,comparable to what is observed in E. coli [23]. The SpoT deficiencycould be complemented in strain χ10019 (ΔrelA233 ΔspoT85 ΔlacZ516::TTaraC P_(BAD) spoT) by the addition of arabinose. Synthesis of SpoT instrain χ10019 in the presence of 0.05% arabinose was nearly identical towild-type SpoT synthesis (FIGS. 3 and 4). The addition of arabinose tostrain χ10019 also restored ppGpp synthesis when cells were starved forcarbon (FIG. 2B).

A cursory examination of the Y. pestis ΔrelA ΔspoT double mutant aftergrowth on solid rich medium indicated that the ΔrelA ΔspoT doublemutants grew more slowly than wild-type or ΔrelA mutants. When growthwas assessed in liquid medium, the ΔrelA ΔspoT mutants exhibited alonger lag phase and did not reach as high a final OD₆₀₀ than thewild-type and ΔrelA mutant strains at both 26° C. and 37° C. (FIGS. 5Aand B). The ΔrelA ΔspoT strains were prone to autoaggregate andprecipitate to the bottom of the culture tube at 26° C., but not at 37°C. The addition of 0.05% arabinose restored wild-type growthcharacteristics to strain χ10019 (ΔrelA233 ΔspoT85 ΔlacZ516::TT araCP_(BAD) spoT) (FIG. 5), but it continued to autoaggregate andprecipitate at 26° C. However, the addition of higher concentrations ofarabinose reduced autoaggregation in a concentration-dependent manner.The addition of 0.4% arabinose resulted in the complete absence ofdetectable autoaggregation at 26° C.

Example 3 The Effect of ppGpp on Production of Virulence Factors of Y.pestis

The virulence of the pathogenic Yersinia species depends on aplasmid-encoded type III secretion system (T3SS) that transfers effectorproteins called Yops (Yersinia outer proteins) into host cells,interfering with mammalian cell signaling pathways, inhibitingphagocytosis, modulating cytokine production, and inducing apoptosis[24]. In S. Typhimurium, pathogenicity islands 1 and 2 (SPI1 and SPI2)encode T3SSs required for invasion and replication within host cells,respectively [25]. SPI1 and SPI2 gene transcription and expression areseverely reduced in the absence of ppGpp [26]. To determine if ppGpp hada similar effect on Y. pestis, transcription of the genes encoding T3SSsubstrates LcrV and Yop proteins was analyzed using RT-PCR. Our resultsindicated that relA or relA spoT status did not have a significanteffect on the transcription of lcrV and or the yop genes (FIG. 6A).

To examine the effect of ppGpp on protein synthesis, the proteome ofwild-type and ΔrelA ΔspoT mutant Y. pestis strains was compared atdifferent temperatures using two-dimensional electrophoresis (FIG. 7).Our results indicate that deletion of relA and spoT led to reducedsynthesis of some metabolic enzymes at flea (26° C.) and human (37° C.)temperatures, and also reduced synthesis of virulence factors such asPla, LcrH and LcrV at 37° C. (Table 1 and Table 2).

TABLE 1 Differentially expressed proteins identified from Y. pestis at26° C. Accession Fold change Protein number Protein name No. FunctionMethod WT/ΔrelAΔspoT 1 PanC (pantoate-beta- y0785 biosynthesis ofcofactors, MALDI 7.3 alanine ligase) carriers: pantothenate 2hypothetical protein y2262 putative MALDI 15.2 3 S-ribosylhomocysteinasey0888 catalyzes the hydrolysis MALDI 8.6 of S-ribosylhomocysteine tohomocysteine and autoinducer-2 4 MetG (methionyl-tRNA y2648 aminoacyltRNA synthetases, MALDI 2.7 synthetase) tRNA modification 5 PyrE(orotate y0096 pyrimidine ribonucleotide MALDI 2.5phosphoribosyltransferase) biosynthesis 6 PyrB (aspartate y0161pyrimidine ribonucleotide MALDI 3.6 carbamoyltransferase biosynthesiscatalytic Subunit)

TABLE 2 Differentially expressed proteins identified from Y. pestis at37° C. Accession Fold change Protein number Protein name No. FunctionMethod WT/ΔrelAΔspoT 1 LcrH (SycD) secretion chaperone YPCD1.30cchaperone for YopBD MALDI 2.3 2 FrsA (fermentation/respiration y0964FrsA may promote MALDI 2.8 switch protein) fermentation 3 MetK(S-adenosylmethionine y3314 catalyzes the formation MALDI 4.2synthetase) of S-adenosylmethionine from methionine and ATP; methionineadenosyltransferase 4 CodA (cytosine deaminase) y3946 salvage ofnucleosides and MALDI 1.5 nucleotides 5 Pla (outer membrane protease)YPPCP1.07 outer membrane protease; MALDI 2.6 involved in virulence inmany organisms 6, 7, 8 LcrV (secreted effector YPCD1.31c functions inneedle complex MALDI 7.3 protein) protein export; Yop secretion andtargeting control protein; important for translocation pore formation 9TrpA (tryptophan synthase y2047 amino acid biosynthesis: MALDI 1.6subunit alpha) Tryptophan 10  TyrS (tyrosyl-tRNA synthetase) y1966aminoacyl tRNA synthetases, MALDI 1.6 tRNA modification 11  hypotheticalprotein y2786 putative membrane protein MALDI 2.3 12  Kbl(2-amino-3-ketobutyrate y0081 Central intermediary metabolism: MALDI 1.7coenzyme A ligase) pool, multipurpose conversions

We also evaluated secretion of LcrV and some of the Yops. Recovery ofsecreted Yop proteins is hampered by degradation due to Pla activity[27]. Therefore, secretion of virulence factors was evaluated in Δpladerivatives, χ10023(pCD1Ap) (Δpla), χ10024(pCD1Ap) (Δpla ΔrelA),χ10025(pCD1Ap) (Δpla ΔrelA ΔspoT) and χ10026(pCD1Ap) (Δpla ΔrelA ΔspoTaraC P_(BAD) spoT). The results indicate that LcrV and YopM secretionwas reduced slightly in absence of ppGpp (ΔrelA ΔspoT), but secretion ofYopH, YopD and YopE were significantly decreased (FIG. 6B).

Example 4 A ΔRelA ΔSpoT Mutant is Attenuated in Mice

To investigate the contribution of ppGpp to the virulence of Y. pestis,we infected groups of three Swiss Webster mice subcutaneously withwild-type, χ10003(pCD1Ap) (ΔrelA233), χ10004(pCD1Ap) (ΔrelA233 ΔspoT85)and χ10019(pCD1Ap) (ΔrelA233 ΔspoT85 ΔlacZ516::TT araC P_(BAD) spoT), inwhich spoT expression is regulated by arabinose availability. Strainχ10019(pCD1Ap) was grown in the presence of arabinose prior toinoculation of mice. Once this strain colonizes host tissues where thereis no free arabinose [4], it will become phenotypically SpoT⁻. Inpreliminary experiments we determined that the LD₅₀ of the wild-typestrain in mice is, <10 CFU, consistent with previous results [28,29].Mice given wild-type Y. pestis KIM5+ and χ10003(pCD1Ap) (ΔrelA)succumbed to the infection in a highly synchronous manner (FIG. 8). Only50% of the mice infected with 5.8×10⁵ CFU of ΔrelA ΔspoT strain χ10004developed plague after 6 days, and the rate at which the mice died wasslower than the rate of those infected with the wild-type strain. TheLD50 of χ10004(pCD1Ap) was 5.8×10⁵ CFU. Thus, the lack of ppGpp resultedin a ˜100,000-fold increase in the LD₅₀ obtained by subcutaneous (s.c.)infection. The LD₅₀ of χ10019(pCD1Ap) strain, administered after growthin arabinose was intermediate, at 3.3×10² CFU (˜100-fold increase). TheLD₅₀ of χ10019 (pCD1Ap) was the same as KIM5+ (LD50<10) when inoculatedmice were injected with arabinose, indicating full complementation ofthe attenuation phenotype.

To further evaluate the ability of Y. pestis to disseminate to thebloodstream and internal organs, we monitored the growth of both Y.pestis KIM5⁺ and χ10004(pCD1Ap) in the lungs, spleens, livers and bloodof infected mice over a 7-day period after s.c. injection. Because ofthe difference in LD₅₀ between the two strains, we inoculated mice withdifferent doses of each, 1.5×10³ CFU of Y. pestis KIM5+ or 1.6×10⁶ CFUof χ10004(pCD1Ap). The kinetics of colonization was similar for bothstrains (FIG. 9). Despite the difference in dose, the levels of bacteriain blood, spleen and liver were similar for both strains on days 3 and5. There was an approximate 1.5 log difference in bacteria isolated fromlung tissue, indicating that the ΔrelA ΔspoT mutant was less efficientthan KIM5+ at reaching the lungs. By day 7, the number of the ΔrelAΔspoT mutant began to decline in all tissues, indicating clearance bythe host, while all of the mice inoculated with wild-type Y. pestis hadsuccumbed to the infection.

Example 5 The Immune Responses to ΔrelA ΔspoT Y. pestis Strainχ10004(pCD1Ap)

Because χ10004 was attenuated, we explored its potential as a vaccine.To evaluate the immune responses to ΔrelA ΔspoT Y. pestis strainχ10004(pCD1Ap), two groups of 10 mice each were immunized s.c. with2.5×10⁴ CFU on day 0. Two groups of 4 mice each were injected with PBSas controls. Mice were challenged on day 35 with either 1.5×10⁵ (s.c.)or 2.0×10⁴ (i.n.) CFU of Y. pestis KIM5+. Blood was taken at 2 and 4weeks post immunization and 2 weeks after challenge. Serum IgG responsesto Y. pestis whole cell lysates (YpL) from immunized mice were measuredby ELISA (FIG. 10A). At two weeks after immunization, the reciprocalanti-Y. pestis serum IgG titers were greater than 1,000 and increased at4 weeks and after challenge.

The serum immune responses to YpL were further examined by measuring thelevels of IgG isotype subclasses IgG1 and IgG2a. Th1 cells directcell-mediated immunity and promote class switching to IgG2a, and Th2cells provide potent help for B-cell antibody production and promoteclass switching to IgG1 [30]. The level of anti-YpL IgG1 and IgG2aisotype antibodies rapidly increased after vaccination and graduallyincreased at 2 weeks, 4 weeks and post-challenge (FIG. 10B). At 2 and 4weeks post-immunization, the ratio of IgG1 to IgG2a was 1.06:1 and 1.2:1respectively, indicating an initial mixed Th1/Th2 response, whichdeveloped into a slight Th2 bias by week 4. This Th2 bias continuedafter challenge as well.

Example 6 Immunization with a ΔRelA ΔSpoT Y. Pestis Strainχ10004(pCD1Ap) can Protect Against Plague Challenge

To evaluate the protective efficacy of ΔrelA ΔspoT Y. pestis strainχ10004(pCD1Ap) against the bubonic and pneumonic forms of plague,immunized mice were challenged on day 35 with either 1.5×10⁵ (s.c.) or2.0×10⁴ (i.n.) CFU of Y. pestis KIM5+. Post-challenge survival wasmonitored for 14 days. A single s.c. vaccination could provide completeprotection against s.c. challenge without any symptoms (FIG. 11A) and60% protection against pulmonary challenge (FIG. 11B). None of the miceimmunized with PBS survived either challenge (FIG. 11).

Example 7 Induction of Cytokines by Y. Pestis KIM5+ and ΔRelA ΔSpoTStrain χ10004(pCD1Ap)

Cytokines are critical to the development and functioning of both theinnate and adaptive immune responses. They are often secreted by immunecells that have encountered pathogens, thereby activating and recruitingadditional immune cells to increase the system's response to thepathogen. Previously, LcrV has been demonstrated to be animmunomodulator (TNF-α and IFN-γ down-regulation and IL-10 induction)both in vivo and in vitro [31,32,33]. Since the synthesis and secretionof LcrV is reduced in the ΔrelA ΔspoT mutant, we compared production ofthree cytokines (IL-10, INF-γ and TNF-α) in mice infected with Y. pestisKIM5+ and χ10004(pCD1Ap). For this experiment, groups of threeSwiss-Webster mice were inoculated via the s.c. route with 1.5×10³ CFUof Y. pestis KIM5+ or 1.6×10⁶ CFU of χ10004(pCD1Ap). A group ofuninfected mice served as controls. Blood was collected via cardiacpuncture 3 and 5 days later for cytokine analysis. Measurementsindicated that levels of IL-10 were higher in the sera of animalsinfected with Y. pestis KIM5+ than that of χ10004(pCD1Ap) (FIG. 12). Thepro-inflammatory cytokines IFN-γ and TNF-α were not detected in serafrom mice inoculated with either strain (data not shown)

Materials and Methods for Examples 1-7

Bacterial Strains, Culture Conditions and Plasmids

All bacterial strains and plasmids used in this study are listed inTable 3. All strains were stored at −70° C. in phosphate-bufferedglycerol. Y. pestis cells were grown routinely at 28° C. on Congo redagar from glycerol stocks and then grown in heart infusion broth (HIB)or on tryptose-blood agar base (TBA) [1]. The chemically defined mediumPMH2 was used routinely [2]. All E. coli strains were grown routinely at37° C. in LB broth [3] or LB solidified with 1.2% Bacto Agar (Difco).

TABLE 3 Bacterial strains and plasmids used in this study. Source orStrains Relevant genotype or Annotation derivation E. coli TOP10 F⁻mcrAΔ (mrr-hsdRMS-mcrBC) φ80lacZΔM15 Invitrogen ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupG Y. pestis KIM6⁺ Pgm⁺, pMT1,pPCP1, cured of pCD1 [2] Y. pestis KIM5⁺ Y. pestis KIM6+ pCD1Ap [2]χ10003 ΔrelA233 Y. pestis KIM6+ [5] χ10004 ΔrelA233 ΔspoT85 Y. pestisKIM6+ [5] χ10019 ΔrelA233 ΔspoT85 ΔlacZ516::TT araC P_(BAD) spoT Thisstudy Y. pestis KIM6+ χ10021 spoT412:: 3xFlag-Kan Y. pestis KIM6+ Thisstudy χ10022 ΔrelA233 ΔspoT85 ΔlacZΩTT araC P_(BAD) spoT413:: This study3xFlag-Kan Y. pestis KIM6+ χ10023 Δpla-525 Y. pestis KIM6+ This studyχ10024 ΔrelA233 Δpla-525 Y. pestis KIM6+ This study χ10025 ΔrelA233ΔspoT85 Δpla-525 Y. pestis KIM6+ This study χ10026 ΔrelA233 ΔspoT85Δpla-525 ΔlacZ516::TT araC This study P_(BAD) spoT Y. pestis KIM6+χ10003(pCD1Ap) ΔrelA233 Y. pestis KIM6+ pCD1Ap This study χ10004(pCD1Ap)ΔrelA233 ΔspoT85 Y. pestis KIM6+ pCD1Ap This study χ10019(pCD1Ap)ΔrelA233 ΔspoT85 ΔlacZ::TT araC P_(BAD)spoT This study Y. pestis KIM6+pCD1Ap χ10023(pCD1Ap) Δpla-525 Y. pestis KIM6+ pCD1Ap This studyχ10024(pCD1Ap) ΔrelA233 Δpla-525 Y. pestis KIM6+ pCD1Ap This studyχ10025(pCD1Ap) ΔrelA233 ΔspoT85 Δpla-525 Y. pestis KIM6+ pCD1Ap Thisstudy χ10026(pCD1Ap) ΔrelA233 ΔspoT85 Δpla-525 ΔlacZ516::TT araC P_(BAD)This study spoT Y. pestis KIM6+ pCD1Ap Plasmids Source pUC18 For cloningand sequencing Invitrogen pCD1Ap 70.5-kb pCD1 with bla cassette insertedinto ′yadA; [2] 71.7-kb Lcr⁺ Ap^(r) pCP20 Ap^(r) Cm^(r), FLP recombinaseexpression [7] pKD3 Ap^(r) Cm^(r), cat cassette template [7] pKD46Ap^(r), λ Red recombinase expression [7] pYA3700 TT araC P_(BAD)cassette plasmid, Ap^(r) US Patent. Publ. 2006-0140975 pSUB11 Kn^(r),3xFlag-tagged [6] pYA4373 The cat-sacB cassette in the PstI and SacIsites of pUC18. pUC18 pYA4573 The lacZ-U (upstream gene sequence oflacZ), and pYA3700 lacZ-D (downstream gene sequence of lacZ) fragmentwere cloned into the SphI/PstI sites and SacI/EcoRI sites of pYA3700respectively. pYA4574 The spoT gene with new SD sequence was cloned intopYA4573 the XhoI and SacI sites of pYA4573. pYA4575 The cat-sacBcassette from pYA4373 was ligated into pYA4574 PstI site of pYA4574.pYA4642 The C-terminal spoT gene fragment (510 bp) was pUC18 cloned intoHindIII and BamHI sites of pUC18. pYA4643 The spoU′ gene fragment(downstream sequence of pYA4642 spoT) was cloned into SacI and EcoRIsites of pYA4642. pYA4644 The lacZ-D gene fragment (downstream sequenceof pYA4642 lacZ) was cloned into SacI and EcoRI sites of pYA4642.pYA4645 The 3xFlag::kan gene fragment was cloned into SacI pYA4643 andBamHI sites of pYA4643. pYA4646 The 3xFlag::kan gene fragment was clonedinto SacI pYA4644 and BamHI sites of pYA4644. pYA4647 The pla-U fragment(upstream sequence of pla) was pUC18 cloned into the EcoRI and PstIsites of pUC18. pYA4648 The pla-D fragment (downstream sequence of pla)pYA4647 was cloned into the SphI and PstI sites of pYA4647. pYA4649 Thecat cassette (including Flp recombination site) pYA4648 was cloned intothe PstI site of pYA4648.Plasmid Construction

All primers used are listed in Table 4. The original source for thetightly regulated araC P_(BAD) in pYA3700 was E. coli K-12 strain χ289[4]. For construction of the P_(BAD) spoT insertion/deletion into lacZ,primer sets of LacZ1/LacZ2 and LacZ3/LacZ4 were used for amplifyinglacZ-U (upstream gene sequence of lacZ), and lacZ-D (downstream genesequence of lacZ) fragment, respectively. The lacZ-U and lacZ-Dfragments were cloned into the SphI/PstI sites and SacI/EcoRI sites ofpYA3700 to form pYA4573. The spoT gene fragment was amplified usingSpoT-1 and SpoT-2 primers. The primer SpoT-1 containing the new SDsequence is shown Table 4. The spoT fragment was cloned into pYA4573 toconstruct pYA4574. Plasmid pYA4574 was digested with PstI, blunt endedwith T4 DNA polymerase and dephosphorylated with shrimp alkalinephosphatase (Promega). The cat-sacB fragment was cut from pYA4373 usingPstI and SacI restriction endonucleases and blunted by T4 DNApolymerase. Then, the cat-sacB fragment was ligated into PstI site ofpYA4574 to form plasmid pYA4575.

TABLE 4 Oligonucleotides used in this work Seq. Name Sequence ID No.LacZ1 ^(a) 5′ cggctgcagcccatcactccagcgcagaact 3′ (PstI) 1 LacZ2 5′cgggcatgctccagcccattcaggcttat 3′ (SphI) 2 LacZ3 5′cgggaattccaaaggagcaatgcatgtatgg 3′ (EcoRI) 3 LacZ4 5′cgggagctccatgtgttgccaactggctg 3′ (SacI) 4 LacZ5 5′ctaaattgttatctcttcgtag 3′ 5 LacZ6 5′ tgcagggagatgagttaacaatg 3′ 6SpoT-1 ^(a,b) 5′ cggctcgag GGAGTGaaacgTTGtacctgtttgaaagcct 3′ 7 (XhoI)SpoT-2 ^(a) 5′ cgggagctcttaattgcgattacggctaactttaacc3′ (SacI) 8 Pla1 5′cgggaattcagcaaaacagacaaacgcctgctgg 3′ (EcoRI) 9 Pla2 5′cggctgcagtagacacccttaatctctctgcatg 3′ (PstI) 10 Pla3 5′cggctgcagtacagatcatatctctcttttcatcctc 3′ (PstI) 11 Pla4 5′cgggcatgcctggtgcgtatagctgaggatgaat 3′ (SphI) 12 Pla5 5′gagataacgtgagcaaaacaaaatctggtcg 3′ 13 Pla6 5′gagccttttatgcgttcgatccgattcg 3′ 14 Cm15′cggaactgcagatgggaattagccatggtcc 3′ (PstI) 15 Cm25′cggctgcagtgtaggctggagctgcttcg 3′ (PstI) 16 SpoTC-1 5′cggaagcttatgagcgtagtggtggctaa 3′ (HindIII) 17 SpoTC-2 5′cggggatccattgcgattacggctaactt 3′ (BamHI) 18 SpoTD-15′cgggagctctaacgcctatgaatcctcaacgctatg 3′ (SacI) 19 SpoTD-2 5′cgggaattctgtgtgtccgtttatacatc 3′ (EcoRI) 20 Flag-1 5′cggggatccgactacaaagaccatgacggtgatt 3′ (BamHI) 21 Flag-2 5′cgggagctccatatgaatatcctccttagttcctat 3′ (SacI) 22 Cm-V5′gttgtccatattggccacgttta3′ 23 SacB-V 5′ gcagaagagatatttttaattgtggacg 3′24 araC-V 5′catccaccgatggataatcgggta3′ 25 16S rRNA 5′aggcgacgatccctagctggtctga 3′ 26 primer1 16S rRNA 5′cgtttacagcgtggactaccagggt 3′ 27 primer2 IcrV primer1 5′tcctagcttattttctacccgagga 3′ 28 IcrV primer2 5′ttaattcggcggtaagctcagctaa 3′ 29 yopB primer1 5′tgtttcagtgctaacgaagtttacgc 3′ 30 yopB primer2 5′acaatcactgaggctatggcgctga 3′ 31 yopD primer1 5′tcttgttgttgctgttggaactggc 3′ 32 yopD primer2 5′gttgttcgcggccagcaatattact 3′ 33 yopE primer1 5′catttgctgcctgcgttagatcaac 3′ 34 yopE primer2 5′gccaaaatacatgcagcagttgaat 3′ 35 yopH primer1 5′tcgtcaggtatctcgattggtgcag 3′ 36 yopH primer2 5′ccattgccgacacttcttaagtcat 3′ 37 yopJ primer1  5′tcacgtatggatgtagaagtcatgc 3′ 38 yopJ primer2  5′gtttttgtccttattgccagcatcg 3′ 39 yopK primer1  5′gtgctttatgtaccgctcttgaaca 3′ 40 yopK primer2  5′gtcaatatcgctgacatgttgccat 3′ 41 yopM 5′ acgtcattcttctaatttaactgagatg 3′42 primer1 yopM 5′ aagtgatttcaggctctgcggtaat 3′ 43 primer2 yopT primer1 5′ tcaaggatagcgtttaataattgatccag 3′ 44 yopT primer2  5′tttatgtgcacattggatcaggagc 3′ 45 * a: the restriction endonuclease sitesare underlined b: the bold capital letters show the Shine-Dalgarno (SD)sequence and the TTG start codon

To construct a spoT-3×-flag-kan fusion, a C-terminal spoT gene fragment(510 bp) was amplified using SpoTC-1 and SpoTC-2 primers and cloned intoHindII and BamHI sites of pUC18 to construct pYA4642. The spoU′ genefragment (sequence downstream of spoT) and lacZ-D gene fragment(sequence downstream of lacZ) were amplified from genomic DNA usingSpoTD-1/SpoTD-2 and LacZ3/LacZ4 primers, respectively. The spoU′ andlacZ-D fragment were cloned into SacI and EcoRI sites of pYA4642 to formpYA4643 and pYA4644, respectively. Then the 3× flag-kan gene fragmentamplified from pYA4045 was cloned into SacI and BamHI sites of pYA4643and pYA4644 to construct pYA4645 and pYA4646.

To delete the pla gene from plasmid pPCP1, plasmids pYA4647, pYA4648,and pYA4649 were constructed. The pla-U fragment was amplified fromtotal DNA of Y. pestis KIM6+ using Pla1 and Pla2 primers and cloned intothe EcoRI and PstI sites of pUC18 to form pYA4647. The pla-D fragmentwas amplified using Pla3 and Pla4 primers. The pla-D fragment was clonedinto pYA4647 to construct pYA4648. The cat cassette (including Flprecombination site) amplified using Cm1 and Cm2 primers was cloned intothe PstI site of pYA4648 to form pYA4649.

Construction of Y. Pestis Mutant Strains

The construction of strains χ10003 and χ10004 using a two-steprecombination method was previously described [5]. Strain x10019 wasconstructed from strain χ10004 using similar methods. Briefly, plasmidpKD46 was introduced into χ10004 by electroporation. A linearlacZ-U-cat-sacB-TT araC P_(BAD) spoT-lacZ-D fragment was purified fromplasmid pYA4575 by digestion with EcoRI and SphI and transformed intoχ10004 (pKD46) competent cells. Electroporants were isolated on TBA+Cm(10 μg/ml) plates. Integration of the lacZ-U-cat-sacB-TT araC P_(BAD)spoT-lacZ-D fragment into the correct site of the chromosome wasverified by PCR. Colonies with the correct PCR profile were streakedonto TBA+Cm (10 μg/ml)+5% Sucrose plates to verify sucrose sensitivityand onto HIB Congo Red+Cm (10 μg/ml) plates to confirm the presence ofthe pgm locus. To remove the cat-sac cassette from the chromosome,electrocompetent cells were prepared from a sucrose-sensitive isolateand electroporated with approximately 1 μg of a linear DNA (lacZ-U-TTaraC) cut from pYA4574 using SphI and BamHI. Electroporants wereselected on TBA+5% sucrose plates incubated at 30° C. Colonies weretested using PCR to validate that the cat-sacB cassette was eliminated.Plasmid pKD46 was cured from a single colony isolate of asucrose-resistant, chloramphenicol-sensitive strain to yield χ10019.

To construct strains expressing spoT tagged with the Flag epitope [6],plasmid pKD46 was introduced into Y. pestis KIM6+ and _(X)10019. Theresulting strains were electroporated with, ˜0.5 μg of spoTC-3×flag-kan-spoU′ and spoTC-3× flag-kan-lacZ-D cut from pYA4645 andpYA4646, respectively. Electroporants were selected on TBA+Kan (20μg/ml) plates at 37° C. The resulting colonies were verified using PCRto confirm that the 3× flag-kan fragment was correctly inserted into thechromosome. Plasmid pKD46 was cured from single colony isolates of Y.pestis KIM5+ or χ10019 derivatives to yield χ10021 and χ10022,respectively.

To construct Pla⁻ mutants, Y. pestis KIM6+ (pKD46), χ10003 (pKD46),χ10004 (pKD46) and _(X)10019 (pKD46) competent cells were electroporatedwith ˜0.5 μg of PCR amplified, gel purified pla-U::cat:pla-D fragmentobtained with primers Pla1 and Pla4 using plasmid pYA4649 as thetemplate. Electroporants were selected on TBA+Cm (10 μg/ml) plates andwere subsequently verified by PCR to confirm that pla was deleted.Plasmid pCP20 was introduced into the pla mutant strains and the Cm^(R)cassette was removed by flip recombinase [7]. Plasmid pCP20 was curedfrom resulting single colony isolates to yield χ10023, χ10024, χ10025and χ10026. Then, the pCD1Ap plasmid was transformed into Y. pestisKIM6+, χ10003, χ10004, χ10019, χ10023, χ10024, χ10025 and χ10026,respectively to form Y. pestis KIM5+, χ10003(pCD1Ap), χ10004(pCD1Ap),χ10019(pCD1Ap), χ10023 (pCD1Ap), χ10024(pCD1Ap), χ10025(pCD1Ap) andχ10026 (pCD1Ap) under BSL3 containment.

ppGpp Assay

ppGpp was detected using a slight modification of previously describedprocedures [8,9]. To starve cells for amino acids, strains were grownovernight in HIB medium at 26° C. The cells were then harvested andwashed three times with PBS and resuspended to an OD₆₂₀ of 0.15 in 1 mlof modified PMH2 medium lacking L-phenylalanine [9]. The culture wasshaken at 250 rpm at 26° C. for approximately 5 h until the OD₆₂₀reached 0.25, whereupon, [³²P] H₃PO₄ was added to 100 μCi/ml. Cells wereincubated for an additional 1 h at 26° C. Following incubation, an equalamount of chilled 90% formic acid was added to the cell suspension. Theice-cold suspensions were then rigorously vortexed followed by threefreeze-thaw cycles. The acid extracts were centrifuged in a minifuge setat the highest speed for 5 min, and 5 ml of supernatant was then appliedto a polyethyleneimine-cellulose thin-layer chromatography plate (TLC).The TLC plates were developed at room temperature with 1.5 M KH₂PO₄ (pH3.4). The developed plates were then air-dried and visualized byautoradiography using X-ray film at −70° C. To starve cells for carbon,strains were grown overnight in HIB medium. For strain χ10019, twocultures were grown, one with and one without the addition of 0.05%arabinose. The cells were harvested, washed three times using PBS andresuspended to an OD₆₂₀ of 0.15 in 1 ml of modified PMH2 medium withoutglucose or arabinose. Cultures were grown, labeled and evaluated by TLCas described above.

Analysis of Virulence Factor Transcription by RT-PCR

Total RNA was extracted from bacterial cells using TRIzol Reagent(Invitrogen) according to the manufacturer's recommendations. RNAsamples were treated with DNase I for 10 min at 37° C. to degradecontaminating DNA followed by inactivation of DNase I with 2 mM EDTA andheating to 65° C. for 10 min. RNA was then precipitated with sodiumacetate and ethanol and washed with 70% ethanol prior to performingRT-PCR. RNA samples of 200 ng were used for reverse transcription, usingrandom hexamer primers and Superscript II reverse transcriptase asdescribed by the manufacturer (Invitrogen). PCR amplification wasperformed using the lcrV yopB, yopD, yopE, yopH, yopJ, yopK, yopM, yopTor 16S rRNA primer pairs listed in Table 4. RNA samples were used astemplates in PCR reactions for RT minus controls. Twenty cycles ofamplification were performed using an annealing temperature of 58° C.Products were then separated on a 1% agarose gel, stained with ethidiumbromide and imaged for visualization of appropriately sized PCRproducts. In all cases, reactions were performed in triplicate.

Protein Analysis

Secreted virulence factors were prepared by using a modification ofpreviously described methods [10]. Y. pestis was grown in HIB mediumovernight at 26° C. The cells were then harvested and washed three timesusing PMH2, inoculated to 40 ml of fresh PMH2 medium to an OD₆₀₀ of 0.05and shaken overnight at 26° C. Cultures were shifted to 37° C. for 6 hwith shaking to provide mild aeration. Bacterial cells were removed bycentrifugation for RNA extraction. Secreted virulence factors from theculture supernatants were concentrated by precipitation with 10% (w/v)trichloroacetic acid overnight at 4° C. Precipitated proteins werecollected by centrifugation, washed with ice-cold acetone, and dissolvedin 0.05 M Tris-HCl buffer (pH 9.5). Insoluble materials were removed bycentrifugation at 12 500 g for 15 min and the protein concentration inthe supernatant was determined using the DC protein assay kit (Bio-RadLaboratories, Hercules, Calif.). Samples containing 200 μg proteins wereheated at 95° C. for 5 min in protein sample buffer containing2-mercaptoethanol and analyzed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) with 10% polyacrylamide. Proteins weretransferred to nitrocellulose membranes. The membranes were blocked with5% skim milk in PBS, incubated with rabbit polyclonal antibodiesspecific for the indicated Yop proteins or LcrV, and washed withPBS-Tween 20. Then alkaline phosphatase-conjugated goat anti-rabbitimmunoglobulin G (IgG) (Sigma, St. Louis, Mo.) was added in PBS-Milk.Immunoreactive bands were detected by the addition of NBT/BCIP (Sigma,St. Louis, Mo.). The reaction was stopped after 5 min by washing withseveral large volumes of deionized water.

Two-Dimensional Gel Electrophoresis

Comparison of two dimensional protein profiles was carried out aspreviously described [11]. Y. pestis KIM5+ and χ10004(pCD1Ap) were grownat 26° C. or 37° C. in 5 ml of best-case-scenario (BCS) medium withoutCa2⁺. The cultures were harvested by centrifugation and washed once withlow salt PBS (0.1×). Cells were resuspended in 1 ml lysis buffercontaining 8M Urea, 0.05M DTT, 2% (w/v) CHAPS and 0.2% (w/v) ampholytes.Proteins were extracted by vortexing 1 ml cell samples in lysis bufferwith 0.2 mm glass beads ten times for 30 s with cooling betweenvortexing. The samples were centrifuged at 2500 g for 5 min to removethe beads. The bead-free supernatant was centrifuged at 15000 g for 15min at 4° C. to remove cellular debris. The cell-free lysates wereimmediately placed on ice and protease inhibitor was added. The lysateswere retreated with a 2D protein cleanup kit (Bio-Rad, Hercules, Calif.)and protein concentration was determined using the Bio-Rad Protein Assaykit.

Protein lysates (300 μg) were mixed with rehydration buffer (Bio-Rad) ina total volume of 300 μl. Equal amounts (300 μg) of protein wereisoelectrically focused using 17 cm pH 4-7 strips followed by 18.3×19.3cm 8-16% SDS-PAGE using Midi-Protean II 2D cell (Bio-Rad). Gels werestained with Coomassie Brilliant Blue R-250 (Bio-Rad) and visualizedusing Gel Doc XR system (Bio-Rad). Protein expression levels fromprotein spots on gels were compared between the different samples. Gelanalysis was performed using the PDQuest3 2-D Analysis Software(Bio-Rad) to determine differential expression. Differentially expressedprotein spots were excised and were digested with In-Gel TrypticDigestion Kit (Pierce, Rockford, Ill.). Peptide digests were analyzedusing a Voyager DE STR MALDI-TOF mass spectrometer (Applied Biosystems,Framingham, Mass.). Data were searched in bacterial proteomics databaseusing Aldente in ExPASy Proteomics Server. This experiment was performedfour times with similar results.

Virulence Studies in Mice

Single colonies of each strain were used to inoculate HIB cultures andgrown overnight at 26° C. To select for plasmid pCD1Ap, ampicillin wasadded into the medium at a concentration of 25 μg/ml. Bacteria werediluted into 10 ml of fresh HIB enriched with 0.2% xylose and 2.5 mMCaCl₂ to obtain an OD₆₂₀ of 0.1 and incubated at 26° C. for s.c.infections (bubonic plague) or at 37° C. for intranasal (i.n.)infections (pneumonic plague). Both cultures were grown to an OD₆₂₀ of0.6. The cells were then harvested and the pellet resuspended in 1 ml ofisotonic PBS. All animal procedures were approved by the Arizona StateUniversity Animal Care and Use Committee. Female 7-week-old SwissWebster mice from Charles River Laboratories were inoculated by s.c.injection with 100 ml of bacterial suspension. Actual numbers ofcolony-forming units (CFU) inoculated were determined by plating serialdilutions onto TBA agar. To determine 50% lethal dose (LD₅₀), fivegroups of six mice were infected with serial dilutions of the bacterialsuspension. For in vivo complementation of strain of χ10019(pCD1Ap), 120mg of L-arabinose dissolved in PBS was intraperitoneally administered tomice on the day of inoculation and once a day thereafter [12]. Mice weremonitored twice daily for 21 days, and the LD₅₀ was calculated asdescribed [13].

For colonization/dissemination analysis, 3 mice per time point wereinfected by s.c. injection in the front of the neck. At the indicatedtimes after infection, mice were euthanized, and samples of blood,lungs, spleen and liver were removed. The bacterial load for each organwas determined by plating dilutions of the homogenized tissues onto TBAwith ampicillin plates and reported as CFU per gram of tissue or CFU perml blood. Infections were repeated in at least two independentexperiments.

Preparation of Bacterial Antigens

Bacterial antigens used for ELISA were prepared from fresh cells.Briefly, single colonies of Y. pestis KIM5+ were inoculated into HIBmedia and cultured overnight at 26° C. Cells were switched to 37° C. for6 h. Bacterial cultures were centrifuged at 5,000×g for 10 min, thepellet was washed once with sterile PBS and resuspended in sterile PBS.Bacterial cells were broken using 0.2 mm glass beads 10 times for 60 swith cooling between vortexing (with 2 min incubation on ice betweencycles). The whole bacterial lysate was sterilized by UV light andsterility was confirmed by TBA agar culture. The lysate was frozen at−80° C. until use. Protein content was determined by BCA analysis permanufacturer's instructions (Sigma).

Enzyme-Linked Immunosorbent Assay (ELISA)

Mice were lightly anesthetized using ketamine and xylazine mixtureadministered intramuscularly. Blood was collected by retro-orbital sinuspuncture for the determination of antibody titers at different timepoints. ELISA was used to assay serum antibodies against the whole celllysate of Y. pestis KIM5+. Sera were tested for IgG at a startingdilution of 1:1000, and for IgG1 and IgG2a at 1:100, respectively.

Polystyrene 96-well flat-bottom microtiter plates (Dynatech LaboratoriesInc., Chantilly, Va.) were coated with 200 ng/well of Y. pestis wholecell lysates. Antigens suspended in sodium carbonate-bicarbonate coatingbuffer (pH 9.6) were applied in 100 μl volumes to each well. The coatedplates were incubated overnight at 4° C. Free binding sites were blockedwith a blocking buffer (phosphate-buffered saline [PBS; pH 7.4], 0.1%Tween 20, and 1% bovine serum albumin). A 100 μl volume of seriallydiluted sample was added to individual wells in triplicate and incubatedfor 1 h at 37° C. Plates were treated with biotinylated goat anti-mouseIgG, IgG1, or IgG2a (Southern Biotechnology Inc., Birmingham, Ala.).Wells were developed with streptavidin-horseradish peroxidase conjugate(Invitrogen, Carlsbad, Calif.), followed by2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) (Sigma) insodium citrate buffer containing 0.03% hydrogen peroxide (H₂O₂). After a10 min incubation at 37° C. in the dark, color development (absorbance)was recorded at 405 nm using an automated ELISA plate reader (modelEL311SX; Biotek, Winooski, Vt.). Absorbance readings that were 0.1higher than PBS control values were considered positive.

In Vivo Cytokine Analysis

Cytokines were quantitated by a double-sandwich enzyme-linkedimmunosorbent assay (ELISA) as described previously [33]. Mice in groupsof three were euthanized at intervals by terminal bleeding underanesthesia. Pooled blood was allowed to clot overnight at 4° C., andserum was separated by centrifugation at 10,000 g for 10 min. Sera werefiltered once through a 0.22 μm syringe filter, cultured on TBA toconfirm that bacteria had been removed and stored at −70° C. prior toassay for cytokines.

Commercial solid-phase enzyme immunoassays utilizing themultiple-antibody sandwich principal were used to determine cytokines inbiological samples. In these experiments, IL-10, TNFα and IFN-γ weredetermined with Mouse IL-10, IFN-γ and TNF-α Ready-SET-Go kits(ebioscience), respectively. Concentrations of cytokines were measuredby reading optical density at 450 nm and then calculated in reference tovalues obtained in standard curves generated for each assay. Assays ofpooled sera were repeated three times.

Protective Efficacy

Two groups of Swiss Webster mice (10 mice/group) were immunized by s.c.injection with 2.5×10⁴ CFU of χ10004 (pCD1Ap) cells in 100 μl ofisotopic PBS on day 0. Two groups of mice (4 mice/group) were injectedwith 100 μl of PBS as controls. On day 35, animals were challenged bys.c. injection with 100 μl of virulent Y. pestis KIM5+ or lightlyanesthetized with a 1:5 xylazine/ketamine mixture and challenged by theintranasal route with 20 μl of bacterial suspension. The challenge dosefor s.c. injection was 1×10⁵ CFU and for i.n. challenge was 2.0×10⁴ CFU.Protective efficacy was determined by the number of surviving animals.All infected animals were observed over a 15-day period for thedevelopment of signs of plague infection.

Statistical Analysis

Data are expressed as means±SE. One-way analysis of variance withStudent t-test was used for statistical analysis. A P-value of <0.05 wasconsidered significant.

References for Examples 1-7

-   1. Straley S C, Bowmer W S (1986) Virulence genes regulated at the    transcriptional level by Ca²⁺ in Yersinia pestis include structural    genes for outer membrane proteins. Infect Immun 51: 445-454.-   2. Gong S, Bearden S W, Geoffroy V A, Fetherston J D, Perry R    D (2001) Characterization of the Yersinia pestis Yfu ABC inorganic    iron transport system. Infect Immun 69: 2829-2837.-   3. Bertani G (1951) Studies on lysogenesis. I. The mode of phage    liberation by lysogenic Escherichia coli. J Bacteriol 62: 293-300.-   4. Kong W, Wanda S Y, Zhang X, Bollen W, Tinge S A, et al. (2008)    Regulated programmed lysis of recombinant Salmonella in host tissues    to release protective antigens and confer biological containment.    Proc Natl Acad Sci USA 105:9361-9366.-   5. Sun W, Wang S, Curtiss R, 3rd (2008) Highly efficient method for    introducing successive multiple scarless gene deletions and    markerless gene insertions into the Yersinia pestis chromosome. Appl    Environ Microbiol 74: 4241-4245.-   6. Uzzau S, Figueroa-Bossi N, Rubino S, Bossi L (2001) Epitope    tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci USA    98: 15264-15269.-   7. Datsenko K A, Wanner B L (2000) One-step inactivation of    chromosomal genes in Escherichia coli K-12 using PCR products. Proc    Natl Acad Sci USA 97: 6640-6645.-   8. Sarubbi E, Rudd K E, Xiao H, Ikehara K, Kalman M, et al. (1989)    Characterization of the spoT gene of Escherichia coli. J Biol Chem    264:15074-15082.-   9. Charnetzky W T, Brubaker R R (1982) RNA synthesis in Yersinia    pestis during growth restriction in calcium-deficient medium. J    Bacteriol 149: 1089-1095.-   10. Zahorchak R J, Brubaker R R (1982) Effect of exogenous    nucleotides on Ca²⁺ dependence and V antigen synthesis in Yersinia    pestis. Infect Immun 38: 953-959.-   11. Chromy B A, Choi M W, Murphy G A, Gonzales A D, Corzett C H, et    al. (2005) Proteomic characterization of Yersinia pestis virulence.    J Bacteriol 187:8172-8180.-   12. Loessner H, Endmann A, Leschner S, Westphal K, Rohde M, et    al. (2007) Remote control of tumour-targeted Salmonella enterica    serovar Typhimurium by the use of L-arabinose as inducer of    bacterial gene expression in vivo. Cell Microbiol 9: 1529-1537.-   13. Reed L J, Muench H (1938) A simple method of estimating fifty    percent endpoints. Am J Hyg 27: 493-497.-   14. Sheehan K C, Ruddle N H, Schreiber R D (1989) Generation and    characterization of hamster monoclonal antibodies that neutralize    murine tumor necrosis factors. J Immunol 142: 3884-3893.-   15. Deng W, Burland V, Plunkett G 3rd, Boutin A, Mayhew G F, et    al. (2002) Genome sequence of Yersinia pestis KIM. J Bacteriol 184:    4601-4611.-   16. Blattner F R, Plunkett G 3rd, Bloch C A, Perna N T, Burland V,    et al. (1997) The complete genome sequence of Escherichia coli K-12.    Science 277: 1453-1474.-   17. McClelland M, Sanderson K E, Spieth J, Clifton S W, Latreille P,    et al. (2001) Complete genome sequence of Salmonella enterica    serovar Typhimurium LT2. Nature 413: 852-856.-   18. Aravind L, Koonin E V (1998) The HD domain defines a new    superfamily of metal-dependent phosphohydrolases. Trends Biochem Sci    23: 469-472.-   19. Wolf Y I, Aravind L, Grishin N V, Koonin E V (1999) Evolution of    aminoacyl tRNA synthetases-analysis of unique domain architectures    and phylogenetic trees reveals a complex history of horizontal gene    transfer events. Genome Res 9: 689-710.-   20. Chipman D M, Shaanan B (2001) The ACT domain family. Curr Opin    Struct Biol 11: 694-700.-   21. Battesti A, Bouveret E (2006) Acyl carrier protein/SpoT    interaction, the switch linking SpoT-dependent stress response to    fatty acid metabolism. Mol Microbiol 62: 1048-1063.-   22. Gentry D R, Cashel M (1996) Mutational analysis of the    Escherichia coli spoT gene identifies distinct but overlapping    regions involved in ppGpp synthesis and degradation. Mol Microbiol    19: 1373-1384.-   23. Xiao H, Kalman M, Ikehara K, Zemel S, Glaser G, et al. (1991)    Residual guanosine 39,59-bispyrophosphate synthetic activity of relA    null mutants can be eliminated by spoT null mutations. J Biol Chem    266: 5980-5990.-   24. Viboud G I, Bliska J B (2005) Yersinia outer proteins: role in    modulation of host cell signaling responses and pathogenesis. Annu    Rev Microbiol 59: 69-89.-   25. Brumell J H, Grinstein S (2004) Salmonella redirects phagosomal    maturation. Curr Opin Microbiol 7: 78-84.-   26. Thompson A, Rolfe M D, Lucchini S, Schwerk P, Hinton J C, et    al. (2006) The bacterial signal molecule, ppGpp, mediates the    environmental regulation of both the invasion and intracellular    virulence gene programs of Salmonella. J Biol Chem 281: 30112-30121.-   27. Sodeinde O A, Sample A K, Brubaker R R, Goguen J D (1988)    Plasminogen activator/coagulase gene of Yersinia pestis is    responsible for degradation of plasmid-encoded outer membrane    proteins. Infect Immun 56: 2749-2752.-   28. Une T, Brubaker R R (1984) In vivo comparison of avirulent Vwa⁻    and Pgm⁻ or Pstr phenotypes of Yersiniae. Infect Immun 43: 895-900.-   29. Mehigh R J, Sample A K, Brubaker R R (1989) Expression of the    low calcium response in Yersinia pestis. Microb Pathog 6: 203-217.-   30. Gor D O, Rose N R, Greenspan N S (2003) TH1-TH2: a procrustean    paradigm. Nat Immunol 4: 503-505.-   31. Brubaker R R (2003) Interleukin-10 and inhibition of innate    immunity to Yersiniae: roles of Yops and LcrV (V antigen). Infect    Immun 71: 3673-3681.-   32. Motin V L, Nakajima R, Smirnov G B, Brubaker R R (1994) Passive    immunity to Yersiniae mediated by anti-recombinant V antigen and    protein A-V antigen fusion peptide. Infect Immun 62: 4192-4201.-   33. Nedialkov Y A, Motin V L, Brubaker R R (1997) Resistance to    lipopolysaccharide mediated by the Yersinia pestis V    antigen-polyhistidine fusion peptide: amplification of    interleukin-10. Infect Immun 65: 1196-1203.

Introduction for Examples 8-13

In the Examples below, regulated delayed attenuation technology wasapplied to the crp gene in Y. pestis by constructing a strain in whichcrp expression is dependent on the presence of arabinose, a sugar thatis not present in host tissues [43, 45]. Arabinose is provided during invitro growth so the strain expresses crp, making it fully functional tointeract with host tissues. Once it has invaded host cells, where freearabinose is not available, crp is no longer expressed and the strainbecomes attenuated. We compare the virulence and immunogenicity of theregulated delayed attenuation strain with an isogenic Δcrp deletionstrain of Y. pestis.

Example 8 Crp Synthesis and Growth of Y. Pestis Mutants

We constructed mutant Y. pestis strains χ10010 (Δcrp) and χ10017 (araCP_(BAD) crp) (FIG. 13). In the araC P_(BAD) crp mutant χ10017, crpexpression is dependent on the presence of arabinose. Crp was notdetected in either the Δcrp strain χ10010 or the araC P_(BAD) crp strainχ10017 grown in the absence of arabinose (FIG. 14A). Upon arabinoseaddition, χ10017 synthesized roughly the same amount of Crp as wild-typeY. pestis.

Once we had confirmed that Crp synthesis was arabinose-regulated, wemoved plasmid pCD1Ap into both mutants and examined their growth inliquid media. Strain χ10010(pCD1Ap) and _(X)10017(pCD1Ap) withoutarabinose grew more slowly and did not reach the same final OD₆₂₀ as Y.pestis KIM5+ at 26° C. or 37° C. in HIB medium (FIG. 14B). When 0.05%arabinose was included in the growth medium, χ10017(pCD1Ap) grew at thesame rate as wild type.

Example 9 LcrV Synthesis and Secretion in Y. Pestis KIM5+ and MutantDerivatives

Crp is required for expression of the Ysc type 3 secretion system andother virulence factors in Yersinia and functional loss of crpdiminishes Yop secretion by Y. enterocolitica and Y. pestis (8, 24, 39).However, the effect of a crp mutation on LcrV secretion has not beenreported. Therefore we compared LcrV production in cells andsupernatants from Y. pestis KIM5+, χ10010(pCD1Ap) and χ10017(pCD1Ap). Weobserved no difference in LcrV synthesis in whole cell lysates amongstrains (FIG. 15). There was a reduction in the amount of LcrV detectedin supernatants between the wild type and strains χ10010(pCD1Ap) (Δcrp)and χ10017(pCD1Ap) (araC P_(BAD) crp). Wild-type levels of secreted LcrVwere restored when strain χ10017(pCD1Ap) was grown with 0.05% arabinose(FIG. 15).

Example 10 Virulence of Y. Pestis Mutants in Mice

To investigate the contribution of Crp to Y. pestis virulence, weinfected Swiss Webster mice s.c. with Y. pestis KIM5+, χ10010(pCD1Ap)(Δcrp) or χ10017(pCD1Ap) (araC P_(BAD) crp). Strain χ10017(pCD1Ap) wasgrown in the presence of arabinose prior to inoculation. The LD₅₀ of Y.pestis KIM5+ was <10 CFU, consistent with previous results [57]. TheLD50 of the Δcrp mutant χ10010(pCD1Ap) was >3×10⁷CFU. The LD₅₀ of strainχ10017(pCD1Ap) was 4.3×10⁵CFU and the mean time to death was delayed 2-9days compared to the wild type. The LD₅₀ of χ10017(pCD1Ap) was the sameas KIM5+ (LD₅₀<10) when inoculated mice were injected with arabinose,indicating full complementation of the attenuation phenotype. In apreliminary experiment, we found that both the Δcrp and araC P_(BAD) crpmutants were attenuated when administered by the intranasal route, withLD₅₀s>1×10⁴ CFU. However, mice inoculated with 7-9×10³CFU of eithermutant were not protected from subsequent intranasal challenge with5×10³CFU of KIM5+ (data not shown) and therefore, we did not repeatthose experiments.

We evaluated the ability of the Y. pestis mutants to disseminatesystemically compared to Y. pestis KIM5+ by monitoring, over a 9-dayperiod, the lungs, spleen, liver and blood of groups of mice injectedwith each of the strains. Because of the difference in LD₅₀ among thethree strains, we inoculated mice with different doses of each. For thistype of experiment, we typically choose a dose that is higher than theLD₅₀. However, since we were not able to establish an LD₅₀ value for theΔcrp strain (>1×10⁷CFU), we chose a dose that matched the highest dosefor which we had data. For the araC P_(BAD) crp mutant, we chose a dosethat was 10-fold above the LD₅₀. Thus, mice were inoculated with1.5×10³CFU of Y. pestis KIM5+, 4.2×10⁷CFU of χ10010(pCD1Ap) or3.8×10⁶CFU of χ10017(pCD1Ap). The kinetics of infection was similar forboth mutants. At 3 days post-infection (p.i.), the number of bacteriarecovered from the blood, liver, and spleen were similar for all strains(FIG. 16). About half the number of χ10010 and χ10017 cells wasrecovered from lungs compared to the wild-type strain. The numbers ofmutants recovered from all tissues decreased steadily on days 6 and 9.All mice inoculated with Y. pestis KIM5+ succumbed to the infectionbefore day 9, and therefore we do not include any of those mice in ourfigure for that time point.

Example 11 Evaluation of Protective Immunity

Groups of mice were immunized with a single dose of χ10010(pCD1Ap)(Δcrp), 10017(pCD1Ap) (araC P_(BAD) crp) or Y. pestis KIM5 (Pgm⁻) andchallenged 35 days later. For these experiments, we wanted to use thehighest possible immunizing dose for each strain. We based our decisionon immunizing doses for each strain on the LD₅₀ data, shown above.Therefore, we immunized with a dose of 3×10⁴CFU of 10017(pCD1Ap),3.8×10⁷CFU of 10010(pCD1Ap) or 2.5×10⁷CFU of Y. pestis KIM5 (Pgm⁻),respectively. Our results after challenge show that a single s.c. doseof χ10010(pCD1Ap) or Y. pestis KIM5 (Pgm⁻) provided excellent protectionagainst a 1×10⁷LD₅₀ s.c. challenge (FIG. 17A). A single s.c. dose ofχ10017(pCD1Ap) provided complete protection against a 10,000 LD₅₀ s.c.challenge without any symptoms (FIG. 17B). Immunization with strainχ10010(pCD1Ap) delayed the time of death, but ultimately did not provideprotection against a 100 LD₅₀ i.n. challenge. Immunization with the Y.pestis strain χ10017(pCD1Ap) or the pgm mutant strain KIM5 providedsignificant protection (P<0.001), protecting most of the mice against a100 LD₅₀ i.n. challenge (FIG. 17C). None of the mice immunized with PBSsurvived challenge by either route (FIG. 17).

Example 12 Serum Immune Responses

Serum IgG responses to YpL and LcrV from immunized mice were measured byELISA. High anti-YpL (FIG. 18A) titers were slower to develop for thearabinose-regulated crp mutant, χ10017(pCD1Ap) than for the Δcrp mutant,χ10010(pCD1Ap), but by week 4, the titers were similar. Also by week 4,the anti-LcrV (FIG. 18B) serum IgG titers were somewhat higher in miceimmunized with χ10017(pCD1Ap) than with χ10010(pCD1Ap). Titers againstboth antigens were boosted in mice challenged s.c. No boosting wasobserved in the mice immunized with χ10017(pCD1Ap) after i.n. challenge.

Th1 cells direct cell-mediated immunity and promote class switching toIgG2a, and Th2 cells provide potent help for B-cell antibody productionand promote class switching to IgG1 [41]. The Δcrp strain χ10010(pCD1Ap)elicited a strong Th2 biased response against both antigens, with highIgG1 titers and low IgG2 titers (FIG. 19A, 19B). Strain χ10017(pCD1Ap)induced a more balanced Th1/Th2 response (FIG. 19C, 19D). Challenge didnot have much effect on the IgG1/IgG2a ratios, except for the anti-LcrVresponse in mice immunized with χ10010(pCD1Ap), where the responsebecame more balanced.

Example 13 Induction of Cytokines

Cytokines are critical to the development and functioning of both theinnate and adaptive immune response. They are secreted by immune cellsthat have encountered pathogens, thereby activating and recruitingadditional immune cells to respond to the infection. LcrV is animmunomodulator, inhibiting production of TNF-α and IFN-γ and inducingIL-10 in eukaryotic cells both in vivo and in vitro [35,47,49]. Toevaluate the effect of reduced LcrV secretion in the two mutants (FIG.15), we compared production of IL-10, INF-γ and TNF-α in infected mice.Groups of three Swiss-Webster mice were inoculated s.c. with 1,500 CFUof Y. pestis KIM5+, 4.2×10⁷CFU of χ10010(pCD1Ap), or 3.8×10⁶CFU ofχ10017(pCD1Ap). A group of uninfected mice served as controls. Blood wascollected via cardiac puncture at days 3 and 6 p.i. for cytokineanalysis. We could detect IL-10, but not INF-γ or TNF-α in the sera ofanimals infected with Y. pestis KIM5+, but IL-10 and pro-inflammatoryfactors such as INF-γ and TNF-α were not detected in mice infected withχ10010(pCD1Ap) and χ10017(pCD1Ap) (data not shown).

Materials and Methods for Examples 8-13

Media and Reagents.

Tryptose blood agar (TBA) and heart infusion broth (HIB) were fromDifco. Y. pestis strains were grown in HIB and on HIB Congo red agarplates at 30° C. to confirm the pigmentation (Pgm) phenotype of Y.pestis strains [54]. Ampicillin, chloramphenicol and L-arabinose werefrom Sigma (St. Louis, Mo.). Oligonucleotides were from IDT (Coralville,Iowa). Restriction endonucleases were from New England Biolabs. Taq DNApolymerase (New England Biolabs) was used in all PCR tests. Qiagenproducts (Hilden, Germany) were used to isolate plasmid DNA, gel-purifyfragments or purify PCR products. T4 ligase, T4 DNA polymerase andshrimp alkaline phosphatase (SAP) were from Promega.

Bacterial Strains and Plasmids.

Strains and plasmids used are listed in Table 5. E. coli TOP10 was usedfor plasmid propagation. During screening for mutants, Y. pestis wasgrown on TBA agar plates with added chloramphenicol (10 g/ml) or 5%sucrose. Y. pestis was grown at 30° C. for 24 h with shaking (liquidmedia) or for 48 h (solid media) (47).

TABLE 5 Bacterial strains and plasmids used in this study. Strain orplasmid Source, reference, Strains Relevant genotype or annotation orderivation E. coli TOP10 F⁻ mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15Invitrogen ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL endA1nupG χ6212 asd⁻ DH5α derivative (48) Y. pestis KIM6+ Pgm⁺ pMT1 pPCP1,cured of pCD1 (40) Y. pestis KIM5+ Pgm⁺ pMT1 pPCP1 pCD1Ap (40) Y. pestisKIM5 Pgm⁻ pMT1 pPCP1 pCD1Ap (36) χ10010 Δcrp-18 Y. pestis KIM6+ Thisstudy χ10017 ΔP_(crp21)::TT araC P_(BAD) crp Y. pestis KIM6+ This studyPlasmids Source pUC18 Ap^(r) Invitrogen pKD46 repA101(ts) ori λ Redrecombinase expression plasmid (39) pYA3493 Asd⁺ pBR ori β-lactamasesignal sequence-based (42) periplasmic secretion plasmid pYA3700 TT araCP_(BAD) cassette plasmid, Ap^(r) (38) pYA4373 The cat-sacB cassette inthe PstI and SacI sites of pUC18 (56) pYA4443 The 6xHis tag in theC-terminal of lcrV gene was pYA3493 cloned into EcoRI and HindIII sitesof pYA3493 pYA4579 The y3957′-′y3955 fragment ligated by overlappingpUC18 PCR cloned into EcoRI and HindIII sites of pUC18 pYA4581 TheSD-crp and y3957′ fragments cloned into the pYA3700 XhoI/EcoRI sites andPstI/HindIII sites of pYA3700 pYA4588 The cat-sacB cassette from pYA4373cloned into pYA4581 the PstI site of pYA4581Plasmid Construction.

All primers used are listed in Table 6. Primer sets CRP-1/CRP-2 andCRP-3/CRP-4 were used for amplifying the y3957’ (upstream of crp) and‘y3955 (downstream of crp) fragments, respectively. Complementaritybetween primers CRP-2 and CRP-3 are indicated by bold lettering. The‘y3955 and y3957’ fragments were fused by overlapping PCR using primersCRP-1 and CRP-4. The resulting PCR product was digested with EcoRI andHindII and ligated pUC18 digested with the same enzymes to construct theplasmid pYA4597. Primer sets CRP-5/CRP-6 and CRP-7/CRP-8 were used foramplifying crp containing its original SD sequence (SD-crp), and y3957′(−110 to −660 bp upstream of crp) fragment, respectively. The SD-crp andy3957′ fragments were cloned into the XhoI/EcoRI sites and PstI/HindIIIsites of pYA3700, respectively to form pYA4581. Plasmid pYA4581 wasPstI-digested, blunted by T4 DNA polymerase and dephosphorylated withSAP. The cat-sacB fragment was cut from pYA4373 using PstI and SacIrestriction endonucleases and blunted by T4 DNA polymerase. The twofragments were ligated to form plasmid pYA4588. lcrV encoding aC-terminal 6× His was amplified from pCD1Ap using primers lcrV-1 andlcrV-2 and cloned into the EcoRI and HindIII sites of pYA3493 to formpYA4443.

TABLE 6 Primers used in this study SEQ. NAME SEQUENCE ID NO. CRP-1^(a)5′ cggaagcttgagactgaaaatagcggcga 3′ (HindIII) 46 CRP-2 5′gcgactgcaggctgccgagctcttccctctaaaaaccggcgtta 3′ 47 CRP-3 5′gaagagctcggcagcctgcagtcgctgttatcctctgttgttatcg 3′ 48 CRP-4^(a) 5′cgggaattcctttttgtaaaatagacacg 3′ (EcoRI) 49 CRP-5^(a) 5′cgggaattcttaacgggtgccgtaaacga 3′ (EcoRI) 50 CRP-6^(a) 5′cggctcgaggaggataacagcgaatggtt 3′ (XhoI) 51 CRP-7^(a) 5′cggctgcaggccgaaaggtatagccaaggt 3′ (PstI) 52 CRP-8^(a) 5′cggaagcttctgatagatcaactgcgcgc 3′ (HindIII) 53 CRP-9 5′cgacttcgcgtacctcaaagct 3′ 54 CRP-10 5′ tacataaccggaaccacaaccag 3′ 55Cm-V 5′gttgtccatattggccacgttta3′ 56 SacB-V 5′gcagaagagatatttttaattgtggacg 3′ 57 araC-V 5′catccaccgatggataatcgggta3′58 IcrV-1 cgggaattc atgattagagcctacgaaca (EcoRI) 59 IcrV-2cggaagctttcaatgatgatgatgatggtgtttaccagacgtgtcatctag (HindIII) 60 * a:the restriction endonuclease sites are underlined b: the bold lettersshow the reverse complementary region between CRP-3 and CRP-4Preparation of LcrV Antiserum.

Full length his-tagged LcrV was expressed from E. coli χ6212 (pYA4443)and isolated by nickel chromatography. 150μg of His-tagged LcrV proteinwas emulsified with Freund's complete adjuvant, and injected into NewZealand female rabbits from Charles River Laboratories. The rabbits wereimmunized with two booster injections (in Freund's incomplete adjuvant)at 3 week intervals. Antiserum was collected 1 week after the lastbooster injection.

Strain Construction.

Y. pestis mutant strains χ10010 and χ10017 were constructed using thetwo-step recombination method [56]. The procedure was as follows: Y.pestis KIM6+ (pKD46) was electroporated with the lineary3957′-cat-sacB-TT araC P_(BAD) SD-crp fragment excised from plasmidpYA4588 using EcoRI and Hind III. Electroporants were selected on TBA-Cmplates and verified by PCR. Colonies with the correct PCR profile werestreaked onto TBA-Cm-sucrose plates to verify sucrose sensitivity andonto HIB Congo Red-Cm plates to confirm the presence of the pgm locus.This intermediate strain was used for all further constructions. Toconstruct strain χ10017, the chromosomal cat-sac cassette was removed byelectroporating with 1 g of linear DNA (y3957′-TT araC) cut from pYA4581using HindIII and BamHI. The loss of the cat-sac cassette insucrose-resistant colonies was confirmed by PCR. Strain χ10010 wasconstructed by electroporating the intermediate strain with a linear‘y3955-y3957’ fragment cut from pYA4597 using HindIII and EcoRI todelete the entire crp gene. Plasmid pKD46 was cured from a single colonyisolate of the above strains to yield χ10010 (Δcrp) and χ10017 (araCP_(BAD) crp). Under BLS-3 containment, plasmid pCD1Ap was thenintroduced by electroporation into each, yielding χ10010(pCD1Ap) andχ10017(pCD1Ap).

SDS-PAGE and Immunoblot Analyses.

Secreted proteins were prepared by using a modification of previouslydescribed methods [50]. Y. pestis was grown in HIB medium overnight at26° C. Cells were harvested, washed three times in chemically definedmedium PMH2 [40], used to inoculate 40 ml of fresh PMH2 medium to anOD₆₀₀ of 0.05 and shaken at 26° C. overnight. Cultures were shifted to37° C. for 6 h with mild aeration. The OD₆₀₀ of cultures were measured,and bacterial cell pellets were collected by centrifugation. The pelletswere suspended in SDS loading buffer. The volume of sample buffer wasadjusted based on the OD₆₀₀ to normalize the amount loaded. Cells werelysed by heating at 95° C. for 5 min. Culture supernatants wereconcentrated by precipitation with 10% (w/v) trichloroacetic acidovernight at 4° C. and collected by centrifugation. Pellets were washedwith ice-cold acetone and dissolved in 0.05 M Tris-HCl buffer (pH 9.5).Insoluble materials were removed by centrifugation at 12,500×g for 15min and the soluble protein concentration was determined using a DCprotein assay kit (Bio-Rad, Hercules, Calif.). Samples were heated at95° C. for 5 min and separated by SDS-PAGE and blotted ontonitrocellulose membranes. The membranes were probed with rabbitanti-LcrV antibodies as described [34].

Virulence Analysis in Mice.

All animal procedures were conducted in ABSL-3 containment facilitiesand approved by the Arizona State University Animal Care and UseCommittee. Single colonies of Y. pestis KIM5+ strains to be tested inmice were used to inoculate HIB broth containing 25 μg/ml ampicillin andgrown at 26° C. overnight. Bacteria were diluted into 10 ml of freshmedium with 0.2% xylose and 2.5 mM CaC1₂ to an OD₆₂₀ of 0.1 andincubated at 26° C. for subcutaneous (s.c.) infections (bubonic plague)or incubated at 37° C. for intranasal (i.n.) infections (pneumonicplague) and grown to an OD₆₂₀ of 0.6. The cells were harvested bycentrifugation and suspended in 1 ml of isotonic PBS.

Female 7-week-old Swiss Webster mice from Charles River Laboratorieswere inoculated s.c. with 100 μl of the bacterial suspension. Actualnumbers of colony-forming units (CFU) inoculated were determined byplating serial dilutions onto TBA agar. To determine the 50% lethal dose(LD₅₀), five groups of six mice/group were inoculated i.n. or s.c. withserial dilutions of bacteria. Mice were monitored twice daily for 21days, and the LD₅₀ was calculated as described [52]. For in vivocomplementation of strain χ10017(pCD1Ap), 120 mg of L-arabinosedissolved in 100 μl PBS was intraperitoneally administered to mice onthe day of inoculation and once a day thereafter [46].

For colonization/dissemination analysis, groups of mice were injecteds.c. At the indicated times after infection, 3 mice per strain wereeuthanized, and samples of blood, lungs, spleen and liver were removed.The bacterial load for each organ was determined by plating dilutions ofthe homogenized tissues onto TBA plates containing 25 μg/ml ampicillinand reported as CFU per gram of tissue or CFU per ml blood. Infectionswere performed in at least two independent experiments.

Determination of Protective Efficacy.

Y. pestis strains were grown as described above. Two groups of SwissWebster mice (10/group) were vaccinated s.c. with 3.8×10⁷CFU ofχ10010(pCD1Ap) or 3×10⁴CFU of _(X)10017(pCD1Ap) cells in 100 μl of PBSon day 0. Another two groups of mice (4/group) were injected with 100 μlof PBS as controls. Blood was collected by retro-orbital sinus punctureat 2 and 4 weeks post immunization and 2 weeks after challenge forantibody measurement. Mice were lightly anesthetized using a ketamineand xylazine mixture administered intramuscularly before bleeding. Onday 35, animals were challenged s.c. with Y. pestis KIM5+ at either1.3×10⁵CFU for χ10017(pCD1Ap) group or 1.3×10⁷CFU for χ10010(pCD1Ap)group in 100 μl PBS or lightly anesthetized with a 1:5 xylazine/ketaminemixture and challenged i.n. with 1.4×10⁴CFU in 20 μl PBS. Control groupswere challenged with 1.3×10³CFU by both routes. All infected animalswere observed over a 15-day period for the development of signs ofplague infection.

Enzyme-Linked Immunosorbent Assay (ELISA).

ELISA was used to assay serum IgG antibodies against Yersinia whole celllysates (YpL) [55] and purified LcrV antigen of Y. pestis KIM5+.Polystyrene 96-well flat-bottom microtiter plates (Dynatech LaboratoriesInc., Chantilly, Va.) were coated with 200 ng/well of YpL or purifiedLcrV protein. The procedures were same as those described previously[42].

Measurement of Cytokine Concentrations.

Cytokines were quantitated by a double-sandwich enzyme-linkedimmunosorbent assay (ELISA) as described previously [53]. Mice in groupsof three were euthanized at intervals by terminal bleeding underanesthesia. Pooled blood was allowed to clot overnight at 4° C., andserum was separated by centrifugation at 10,000×g for 10 min. Sera werefiltered once through a 0.22 μm syringe filter, cultured on TBA toconfirm that bacteria had been removed and stored at −70° C. prior toassay.

Commercial solid-phase enzyme immunoassays utilizing themultiple-antibody sandwich principal were used to determine cytokines inbiological samples. Levels of IL-10, TNF-α and IFN-γ were determinedwith mouse IL-10, IFN-γ and TNF-α Ready-SET-Go kits (ebioscience),respectively. Concentrations of cytokines were measured by readingoptical density at 450 nm and then calculated in reference to valuesobtained in standard curves generated for each assay. Assays of pooledsera were repeated three times.

Statistical Analysis.

The log rank test was used for analysis of the survival curves. Data areexpressed as means±SE. Student t-test was used for other statisticalanalyses. A P-value of <0.05 was considered significant.

References for Examples 8-13

-   34. Branger, C. G., J. D. Fetherston, R. D. Perry, and    Curtiss, R. III. 2007. Oral vaccination with different antigens from    Yersinia pestis KIM delivered by live attenuated Salmonella    typhimurium elicits a protective immune response against plague. Adv    Exp Med Biol 603:387-399.-   35. Brubaker, R. R. 2003. Interleukin-10 and inhibition of innate    immunity to Yersiniae: roles of Yops and LcrV (V antigen). Infect    Immun 71:3673-3681.-   36. Brubaker, R. R. 1983. The Vwa+ virulence factor of Yersiniae:    the molecular basis of the attendant nutritional requirement for    Ca²⁺. Rev Infect Dis 5 Suppl 4:S748-758.-   37. Cornelis, G. R., A. Boland, A. P. Boyd, C. Geuijen, M.    Iriarte, C. Neyt, M. P. Sory, and I. Stainier. 1998. The virulence    plasmid of Yersinia, an antihost genome. Microbiol Mol Biol Rev    62:1315-1352.-   38. Curtiss, R. III, and Kong, W. 2006. Regulated bacterial lysis    for gene vaccine vector delivery and antigen release. United States    Patent 2006/0140975.-   39. Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation    of chromosomal genes in Escherichia coli K-12 using PCR products.    Proc Natl Acad Sci USA 97:6640-6645.-   40. Gong, S., S. W. Bearden, V. A. Geoffroy, J. D. Fetherston,    and R. D. Perry. 2001. Characterization of the Yersinia pestis Yfu    ABC inorganic iron transport system. Infect Immun 69:2829-2837.-   41. Gor, D. O., N. R. Rose, and N. S. Greenspan. 2003. TH1-TH2: a    procrustean paradigm. Nat Immunol 4:503-505.-   42. Kang, H. Y., J. Srinivasan, and Curtiss, R. III. 2002. Immune    responses to recombinant pneumococcal PspA antigen delivered by live    attenuated Salmonella enterica serovar Typhimurium vaccine. Infect    Immun 70:1739-1749.-   43. Katzman, R. L., E. Lisowska, and R. W. Jeanloz. 1970.    Invertebrate connective tissue. Isolation of D-arabinose from sponge    acidic polysaccharide. Biochem J 119:17-19.-   44. Kim, T. J., S. Chauhan, V. L. Motin, E. B. Goh, M. M. Igo,    and G. M. Young. 2007. Direct transcriptional control of the    plasminogen activator gene of Yersinia pestis by the cyclic AMP    receptor protein. J Bacteriol 189:8890-8900.-   45. Kong, W., S. Y. Wanda, X. Zhang, W. Bollen, S. A. Tinge, K. L.    Roland, and Curtiss, R. III. 2008. Regulated programmed lysis of    recombinant Salmonella in host tissues to release protective    antigens and confer biological containment. Proc Natl Acad Sci USA    105:9361-9366.-   46. Loessner, H., A. Endmann, S. Leschner, K. Westphal, M. Rohde, T.    Miloud, G. Hammerling, K. Neuhaus, and S. Weiss. 2007. Remote    control of tumour-targeted Salmonella enterica serovar Typhimurium    by the use of L-arabinose as inducer of bacterial gene expression in    vivo. Cell Microbiol 9:1529-1537.-   47. Motin, V. L., R. Nakajima, G. B. Smirnov, and R. R.    Brubaker. 1994. Passive immunity to Yersiniae mediated by    anti-recombinant V antigen and protein A-V antigen fusion peptide.    Infect Immun 62:4192-4201.-   48. Nakayama, K., S. M. Kelly, and Curtiss, R., III. 1988.    Construction of an asd⁺ expression-cloning vector: stable    maintenance and high level expression of cloned genes in a    Salmonella vaccine strain. Bio/Technology 6:693-697.-   49. Nedialkov, Y. A., V. L. Motin, and R. R. Brubaker. 1997.    Resistance to lipopolysaccharide mediated by the Yersinia pestis V    antigen-polyhistidine fusion peptide: amplification of    interleukin-10. Infect Immun 65:1196-1203.-   50. Perry, R. D., A. G. Bobrov, O. Kirillina, H. A. Jones, L.    Pedersen, J. Abney, and J. D. Fetherston. 2004. Temperature    regulation of the hemin storage (Hms+) phenotype of Yersinia pestis    is posttranscriptional. J Bacteriol 186:1638-1647.-   51. Petersen, S., and G. M. Young. 2002. Essential role for cyclic    AMP and its receptor protein in Yersinia enterocolitica virulence.    Infect Immun 70:3665-3672.-   52. Reed, L. J., and H. Muench. 1938. A simple method of estimating    fifty percent endpoints. Am. J. Hyg. 27:493-497.-   53. Sheehan, K. C., N. H. Ruddle, and R. D. Schreiber. 1989.    Generation and characterization of hamster monoclonal antibodies    that neutralize murine tumor necrosis factors. J Immunol    142:3884-3893.-   54. Straley, S. C., and W. S. Bowmer. 1986. Virulence genes    regulated at the transcriptional level by Ca²⁺ in Yersinia pestis    include structural genes for outer membrane proteins. Infect Immun    51:445-454.-   55. Sun, W., K. L. Roland, C. G. Branger, X. Kuang, and    Curtiss, R. III. 2009. The role of relA and spoT in Yersinia pestis    KIM5+ pathogenicity. PLoS One 4:e6720.-   56. Sun, W., S. Wang, and Curtiss, R. III. 2008. Highly efficient    method for introducing successive multiple scarless gene deletions    and markerless gene insertions into the Yersinia pestis chromosome.    Appl Environ Microbiol 74:4241-4245.-   57. Une, T., and R. R. Brubaker. 1984. In vivo comparison of    avirulent Vwa- and Pgm- or Pstr phenotypes of Yersiniae. Infect    Immun 43:895-900.

What is claimed is:
 1. A recombinant Yersinia pestis bacterium, whereinthe bacterium comprises the plasmid pCD1, and comprises a araC P_(BAD)crp mutation such that the bacterium has regulated delayed attenuation.2. The recombinant Yersinia pestis bacterium of claim 1, furthercomprising a regulated expression mutation.
 3. A vaccine comprising arecombinant bacterium of claim
 1. 4. The vaccine of claim 3, wherein thevaccine elicits a protective immune response against both pneumonic andbubonic plague.
 5. The vaccine of claim 3, wherein the vaccine elicitsan immune response against Yersinia and at least one additionalpathogen.
 6. The recombinant Yersinia pestis bacterium of claim 1,wherein the bacterium is infectious.
 7. A recombinant Yersinia pestisbacterium, wherein the bacterium comprises: a) pCD1, b) a araC P_(BAD)crp mutation such that the bacterium has regulated delayed attenuation(c) a mutation in relA such that ppGpp synthesis is decreased, and (d) amutation in spoT such that ppGpp synthesis is decreased.
 8. Therecombinant bacterium of claim 7, wherein the bacterium is infectious.9. The recombinant bacterium of claim 1, wherein the bacteriumcomprises: a) pCD1, b) a araC P_(BAD) crp mutation such that thebacterium has regulated delayed attenuation, (c) a mutation in relA suchthat ppGpp synthesis is decreased, and (d) a mutation in spoT such thatppGpp synthesis is decreased.
 10. The recombinant bacterium of claim 1,wherein the bacterium comprises: a) pCD1, b) a araC P_(BAD) crp mutationsuch that the bacterium has regulated delayed attenuation, and c) asecond regulated attenuation mutation, such that the bacterium iscapable of colonizing a host in a non-attenuated manner, and ppGppsynthesis is decreased.
 11. A recombinant bacterium of claim 1, whereinthe bacterium comprises: a) pCD1 b) a relA inactivating mutation, c) aspoT inactivating mutation, d) a ΔlacZ::TT araC P_(BAD) spoT mutation,and e) a araC P_(BAD) crp mutation such that the bacterium has regulateddelayed attenuation.
 12. A method of inducing a protective immuneresponse in a host, the method comprising administering an immunogenicamount of a bacterium of claim 1 to the host.
 13. A method of inducing aprotective immune response in a host, the method comprisingadministering an immunogenic amount of a bacterium of claim 7 to thehost.